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Glycemic and Insulinemic Responses Are Affected by Age of Horse and Method of Feed Processing
ORIGINAL RESEARCH Glycemic and Insulinemic Responses Are Affected by Age of Horse and Method of Feed Processing Brian D. Nielsen, PhD,a Cara I. O’Connor-Robison, MS,a Holly S. Spooner, PhD,b and Jason Shelton, PhDc INTRODUCTION
ABSTRACT The objective of this study was to examine age-related differences in glycemic and insulinemic responses of horses that were fed various feedstuffs, with particular attention to method of feed processing. A 16 16 Latin square design was used with eight 2-year-olds and eight mature Arabians. Horses were maintained on a roughage diet and were subjected to a glycemic response test once weekly. A control treatment consisted of an oral dextrose drench (0.25 g dextrose/kg of BW). Ten treatments consisted of variously processed feed ingredients fed at the rate of 1.5 g/kg of BW. Five other treatments were commercial feeds of a proprietary nature and are not reported. Fasting blood samples were taken once a week for 16 weeks. Thirty minutes later, another baseline sample was taken and horses were administered their respective treatment. Further blood samples were taken every 30 minutes through four hours. Samples were analyzed for glucose and insulin concentrations. Differences in glucose response between 2-year-olds and mature horses were minimal. However, mature horses had a higher insulin response (P < .01) suggesting young horses had greater insulin sensitivity. Additionally, differences (P < .05) existed between treatments with pelleted steam-processed corn having the highest glycemic response and cracked corn the lowest. Results from this study confirm that mature horses have reduced insulin sensitivity and that both glycemic and insulinemic responses are altered with feed processing techniques. Thermal processing produces the greatest response; however, a low glycemic response may not be desirable if starch escapes into the hindgut. Keywords: Feed processing; response; Horse; Insulin
Glucose;
Glycemic
From theDepartment of Animal Science, Michigan State University, East Lansing, MIa; Davis College of Agriculture, Forestry and Consumer Sciences, West Virginia University, Morgantown, WVb; and Cargill Animal Nutrition, Minnetonka, MNc. Reprint requests: Brian D. Nielsen, PhD, Department of Animal Science, Michigan State University, 1287D Anthony Hall, East Lansing, MI 48824. 0737-0806/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2010.03.008
Journal of Equine Veterinary Science Vol 30, No 5 (2010)
Many research studies have been conducted to investigate the glycemic responses of horses to various feedstuffs. The demand for this research has been driven, in part, by the theory that insulin resistance increases the risk of certain diseases, such as laminitis and osteochondrosis,1-4 and such insulin resistance may be affected by the glycemic characteristics of the diet.1 Previous studies have demonstrated that feed type, intake, and feed processing all contribute to varying glycemic responses in horses.5-7 Additionally, the glycemic index (GI) of a meal has been shown to alter glucose use during exercise in horses.8 Although the glycemic and insulinemic responses of various feeds have been examined in weanlings,2,9 the comparison of responses in young horses with that of mature horses is limited. Murphy et al compared the plasma glucose response to an oral glucose tolerance test in both weanling and mature ponies, with lower plasma glucose concentrations being reported in mature ponies.10 However, insulin was not measured. When an identically formulated concentrate was tested in three forms (5-mm extruded, 4-mm pellet, 19-mm pellet), there were minimal differences between glucose and insulin responses, but the concentrate was formulated to be low in sugar and starch.11 Whether the same would be true for more traditional feedstuffs is unclear. Therefore, the hypothesis of this study is that young horses have a lower insulin response than the mature horses when fed various feedstuffs.
MATERIALS AND METHODS Animal Care and Management A 16 16 Latin square design was used to test the response of 15 variously processed feedstuffs and commercial feeds designed for horses, along with an oral dextrose drench that was used as a control. Sixteen Arabian horses, specifically four 2-year-old geldings (414 3 kg; 28 1 month), four 2-year-old fillies (397 3 kg; 28 1 month), four mature geldings (480 2 kg; 14.3 0.5 year), and four mature mares (466 2 kg; 14.1 0.5 year), were maintained on pasture with access to a trace-mineralized salt block. Horses received light exercise either through horsemanship classes (mature horses) or through training classes (2-year-old horses) for approximately 1 hour 3–4 days per
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week for the duration of the study. Horses were supplemented with grass hay but received no concentrate to ensure they were on what would be considered a low glycemic-response diet. Each horse was tested for glycemic and insulinemic response once per week for 16 weeks, with testing done twice a week on eight horses per day. The afternoon before each test, the horses were brought into stalls from their pasture, weighed on a digital scale, and fed 0.5 % BW in timothy hay. All hay provided was consumed by the next morning with no refusals. Horses were kept in their individual stalls until sampling was completed. Sampling was conducted the following morning after a 12-hour fast to normalize blood glucose and insulin concentrations.12 Horses had ad libitum access to water throughout the study. The morning of sampling, each horse was catheterized with a 14-cm, 14-G catheter placed into either the left or the right jugular vein. An extension set with a stopcock was placed onto the catheter and sutured into place. The catheters were in place 30 minutes before the first blood sample, which was typically taken between 07:00 and 08:00 AM. After the final blood draw of each test, the catheters were removed and horses were returned to the pasture. The protocol was approved by the Michigan State University Institutional Animal Care and Use Committee (AUF# 09/06-117-0). Treatments A control treatment consisted of dextrose administered at the dosage of 0.25 g dextrose/kg of BW in a 50% oral drench (with water). Ten treatments consisted of variously processed ingredients used in equine feeds and included whole oats, rolled oats, high fat rice bran, pelleted corn, cracked corn, pelleted steam-processed corn, EnergX [expanded 1], EnergX [expanded 2], EnergX [pelleted], and EnergX [unprocessed], where EnergX is a unique combination of food grade corn particles from the Cargill Dry Corn Ingredient business unit (Paris, IL, USA). The grain used to make EnergX is the result of an identity-preserved system, which starts with the selection of toxin-resistant hybrids that are contract grown and traced through the processing system to deliver a unique, consistent nutrient profile for equine feeds. The difference between the two expanded types of EnergX is that the ‘‘expanded 2’’ process involves an increase in the amount of heat and water used to increase the gelatinization of starch. Although the first type of expanding involves using about 5% steam (added in the conditioner and some on the barrel) and an internal dye head temperature of about 1438C, the second type of expanding involves using about 7.5% steam (again, added in the conditioner and some on the barrel) and an internal dye head temperature of about 1548C. To reduce variation in the corn treatments, the cracked, pelleted, and pelleted steam-processed corn were all made from the same corn source. The five other treatments consisted of various
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commercial feeds. Although used in the 16 16 Latin square design to evaluate potential overall differences in glucose or insulin responses associated with age or gender, the feeds were of a proprietary nature and were composed of various combinations of ingredients as opposed to single ingredients in the other 10 treatments. Hence, the individual means from those five proprietary feeds are not reported. Each of the 16 horses was tested using the control or one of the feedstuffs or feeds each week for 16 weeks in a balanced Latin square design where treatment order was varied from horse to horse to eliminate any potential carryover effects. The feeds or feedstuffs were administered at the rate of 1.5 g/kg of BW on an as-fed basis of their weight from the previous week. The amount of feed consumed within 1 hour of offering was recorded along with the time taken until feed consumption ceased. Any feed not consumed was subtracted from the amount fed to determine the total amount eaten. Sample Handling Feed was processed at Cargill Animal Nutrition Innovation Campus (Elk River, MN, USA) with the expanded products processed at Cargill Dry Corn Ingredients. The feeds were bagged with an identifying code to which the researchers were blinded and then shipped to the Michigan State University Horse Teaching and Research Center. Feed samples were retained for full nutrient analysis by Cargill Animal Nutrition. On arrival at Michigan State University, each feed was sampled for later starch analysis. Samples were stored at 208C until analyzed. Blood samples were taken at 30, 0 (just before administering the appropriate treatment), 30, 60, 90, 120, 150, 180, and 240 minutes after treatment. After each blood sample, the catheter was flushed with 10 mL of heparinized saline (10 U heparin/mL saline). The blood was then injected into vacutainer tubes with potassium oxalate and sodium fluoride for the plasma samples to be used for glucose determination and with no preservative for the serum samples to be used for insulin determination. Tubes were placed on ice until they could be centrifuged at 3,000 g for 10 minutes (plasma tubes) or 3,000 g for 25 minutes (serum tubes). Plasma and serum were stored at 208C until analyzed. Sample Analyses Cargill Animal Nutrition provided nutrient analyses of the various treatments except for total starch (Table 1). Samples were analyzed for the determination of dry matter (DM), crude protein (CP), acid detergent fiber (ADF), neutral detergent fiber (NDF), ether extract (EE), and gelatinized starch (CP, AOAC 968.06; ADF and NDF, AOAC 2002.04/973.18, EE, AOAC 920.39; ash, AOAC 942.05). Gelatinized starch was measured by the methods described by Xiong et al.13 The starch content
1.50 (0.04) 1.48 (0.11) 1.40 (0.21) 0.87 (0.65) 1.51 (0.05)a 1.44 (0.18)a,b 1.46 (0.21)a,b 1.04 (0.61)c
1.53 (0.05) 1.39 (0.24) 1.52 (0.03) 1.23 (0.54)
of the feedstuffs was analyzed at Michigan State University by digesting 0.5 g of sample in duplicate. In brief, samples were digested with 20 mL H2O, 10 mL acetate buffer, 0.8 mL HCl, and 250 mL of Crystalzyme 40 L (Valley Research, South Bend, IN) in a 558C water bath for 16 hours. After the digestion, the liquid was diluted to 200 mL, centrifuged, and then was analyzed for free glucose. Glucose was analyzed using a commercially available kit (Autokit Glucose, Wako Diagnostics, Richmond, VA) that was adapted to 96-well plates. In brief, 9 mL of standard, sample, or control were pipetted onto a 96-well plate. Then, 350 mL of buffer solution was added to each well and incubated at 368C for 18 minutes. The plate was then read on a UV spectrophotometer (SpectraMax 340, Molecular Devices, Sunnyvale, CA) at 505 nm. Insulin was analyzed using a commercially available RIA (Coat-A Count Insulin, Diagnostic Products Corp., Los Angeles, CA) as directed by the package insert. Duplicates were accepted when variation was at or below 5% for glucose and 10% for insulin.
Means of the amount eaten (all horses combined) not sharing a common superscript differ (P < .05). a,b,c,d
8.3 9.6 9.5 10.2 2.98 2.99 3.05 3.14 87.7 90.0 88.8 88.8
12.4 12.7 12.4 13.0
4.38 4.54 3.91 4.17
17.63 18.49 15.86 16.87
43.3 37.3 45.6 37.5
81.1 60.0 31.6 23.4
0.28 (0.06) 1.49 (0.06) 1.50 (0.03) 1.52 (0.02) 1.48 (0.09) 1.26 (0.43) 1.32 (0.50) 5.5 5.9 18.6 2.8 2.9 3.8 3.24 2.15 11.44 1.34 1.17 1.36 89.5 87.3 93.1 86.5 85.7 88.2
15.1 13.2 14.4 8.7 8.5 8.8
12.14 3.24 5.69 2.07 1.79 1.63
27.37 10.17 13.44 8.06 7.07 7.51
28.1 53.2 22.5 67.4 63.5 64.1
26.2 30.7 57.3 42.0 18.7 54.8
0.27 (0.05)d 1.42 (0.35)a,b 1.45 (0.17)a,b 1.29 (0.47)b 1.50 (0.07)a 1.38 (0.33)a,b 1.43 (0.35)a,b
0.27 (0.04) 1.36 (0.48) 1.41 (0.23) 1.09 (0.59) 1.53 (0.03) 1.52 (0.02) 1.53 (0.04)
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Dextrose Whole oats Rolled oats High fat rice bran Pelleted corn Cracked corn Pelleted steamprocessed corn EnergX [expanded 1] EnergX [expanded 2] EnergX [pelleted] EnergX [unprocessed]
Treatment
% Total All Horses 2-Year-Olds Mature Starch, Grams Eaten/ Grams Eaten/ Grams Eaten/ % DM % Ash % CP % Fat % ADF % NDF % Starch Gelatinized kg BW (SEM) kg BW (SEM) kg BW (SEM)
Table 1. Nutrient analyses of the feed ingredients tested and the average amount of each treatment consumed (g/kg BW SEM) on an as-fed basis
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Glycemic and Insulinemic Response Calculations The glycemic and insulin indices were determined by averaging the responses at 30 and 0 minutes pre-feeding for a baseline and then using the trapezoidal method of numerical integration14 to calculate the area under the curve (AUC) above baseline values. Statistical analyses were run on the AUC but to provide comparative indices to the control, the AUC for dextrose was set at 100 and the other treatments were reported relative to that, to determine both a GI and insulinemic index (II). To account for potential differences in the amount consumed, the AUC/g of feed and AUC/g starch eaten were also calculated. The peak plasma glucose and serum insulin concentrations above baseline were identified from the sample points, as were the times until the peak concentrations of both glucose and insulin were reached. Statistics Statistics on the 16 16 Latin square design were performed using SAS 8.2 (SAS Inst., Inc., Cary, NC, USA). PROC MIXED was used to evaluate glucose AUC, glucose AUC divided by grams of treatment consumed, insulin AUC, insulin AUC divided by grams of treatment consumed, and both the glucose and insulin AUC divided by grams of treatment and starch consumed, with day and horse nested in age as the random variables. PROC GLM was used to detect differences in peak glucose concentrations, time to peak glucose concentrations, peak insulin concentrations, and time to peak insulin concentrations. There was no affect of gender, and so it was removed from the model. The dependent variables were age, treatment, and age by treatment. Age was tested by horse nested in age. Treatment by age was tested by the residual. When main effect differences were detected, mean
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separation tests were performed. When the amount eaten on a kg/BW basis was found to be significant for an effect on peak plasma glucose and serum insulin concentrations, it was used as a covariate in the model.
RESULTS The amount of each treatment consumed on a g/kg of BW basis differed (P < .001; Table 1). By design, horses consumed less dextrose than the other tested treatments. Horses consumed less of the EnergX [unprocessed] than they did of the remaining treatments (P % .05). Horses also consumed more pelleted corn and EnergX [expanded 1] than they did of high fat rice bran (P % .05). Mature horses consumed the allotted feed faster (15.9 3.7 minutes) than did the 2-yearolds (33.7 3.9 minutes; P < .001). The GI as determined by the AUC did not differ by age (P ¼ .38) or age by treatment interaction (P ¼ .96). However, the GI of feeds differed (P < .001; Table 2) with rolled oats (GI ¼ 208), pelleted corn (GI ¼ 213), pelleted steamprocessed corn (GI ¼ 278), EnergX [expanded 1] (GI ¼ 170), and EnergX [pelleted] (GI ¼ 156) having a greater GI than the dextrose control (GI ¼ 100; P < .05). There was a trend for EnergX [expanded 2] (GI ¼ 144) to have a greater GI than the control (P ¼ .09). When the AUC for plasma glucose was evaluated on the basis of amount of feed consumed to account for differences in intake, there was no difference in the GI response by age (P ¼ .58) or age by treatment interaction (P ¼ .15). However, there was a treatment difference (P < .001; Table 2). The glucose AUC response on the basis of the amount eaten was greater with dextrose (1.25 mg/dL/min) than the other feed ingredients, which ranged from a high of 0.72 mg/dL/min for pelleted steam-processed corn to a low of 0.18 mg/ dL/min for cracked corn. Peak plasma glucose concentrations differed by age (P ¼ .002) and treatment (P < .001; Table 3). Two-year-old horses had a greater peak plasma glucose concentration (131.4 1.5 mg/dL) than did the mature horses (124.8 1.5 mg/dL). The peak plasma glucose concentration of pelleted steam-processed corn was higher than all other treatments. The lowest peak plasma glucose concentrations were recorded with the whole oats, high fat rice bran, cracked corn, EnergX [pelleted], and EnergX [unprocessed] treatments. Other significant variables in the model included the amount eaten on a kg BW basis, horse(age) and day, all at P < .01. Time to peak plasma glucose concentration was longer overall in 2-year-olds (P < .05) and varied by treatment (P < .05; Table 3). The 2-year-olds reached their peak plasma glucose concentrations at 124 4 minutes, whereas mature horses reached at 112 4 minutes. The shortest time to peak plasma glucose concentration occurred with the oral
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drench of dextrose (76 minutes), whereas the longest occurred with pelleted corn (141 minutes). There were age (mature horses greater than 2-year-olds; P ¼ .002), treatment (P < .001), and age by treatment interaction differences (P ¼ .001) for the average II (Table 4). High fat rice bran (II ¼ 107), cracked corn (II ¼ 111), EnergX [unprocessed] (II ¼ 117), and EnergX [expanded 2] (II ¼ 184) did not differ from the dextrose control (II ¼ 100), but both pelleted corn (II ¼ 372) and pelleted steam processed corn (II ¼ 377) were greater than all other treatments (P < .05). Although serum insulin AUC was greater for the mature horses than for the 2-year-olds (P ¼ .002), the age response is more apparent by examining the AUC for serum insulin (mIU/mL/min) on the basis of grams of treatment consumed (Table 4), in which there were also age (P ¼ .006), treatment (P < .001), and age by treatment interaction differences (P ¼ .04). The mature horses had an average insulin AUC/g consumed of 0.89 0.14 mIU/mL/min/g feed, whereas it was only 0.26 0.14 mIU/mL/min/g feed for the 2-year-olds. The dextrose treatment had the highest AUC/g consumed for insulin, whereas whole oats, rolled oats, high fat rice bran, cracked corn, and the four EnergX treatments had the lowest. For individual treatments, mature horses had greater serum insulin AUC/g consumed for dextrose, whole oats, pelleted corn, pelleted steam-processed corn, EnergX [pelleted], and EnergX [unprocessed]. Differences in peak serum insulin concentrations were detected for age and treatment (both at P < .001) and there was a trend for an age by treatment interaction difference (P ¼ .09; Table 5). Also having an effect on peak serum insulin concentrations were amount eaten per kg of BW (P ¼ .03) and horse(age) (P < .001) with a trend for a day difference (P ¼ .08). The overall peak serum insulin concentration was greater with mature horses (122 4 mIU/mL) than with the 2-year-olds (31 4 mIU/mL; P < .001). Pelleted corn had the greatest peak serum insulin concentration at 135 mIU/mL, with the pelleted steam processed corn being the next highest at 108 mIU/mL. Lowest peak serum insulin concentrations were with the cracked corn (51 mIU/mL), high fat rice bran (61 mIU/ mL), EnergX [unprocessed] (57 mIU/mL), and EnergX [pelleted] (68 mIU/mL). Differences in time to peak insulin concentrations existed for both age (P ¼ .001) and treatment (P ¼ .005; Table 5). As with peak glucose concentrations, peak serum insulin concentrations occurred at a later time with 2-year-olds (123 5 minutes) than with the mature horses (100 5 minutes; P ¼ .001). The shortest time was with the dextrose control (88 minutes), cracked corn (94 minutes), EnergX [expanded 1] (94 minutes), and EnergX [expanded 2] 89 minutes). The longest time was with rolled oats (141 minutes) and EnergX [pelleted] (140 minutes).
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Table 2. Overall mean treatment glycemic indices (GI), the treatment GI by age of horse, overall mean treatment plasma glucose area under the curve (mg/dL/min) divided by the grams of treatment consumed (glucose AUC/g), and the treatment glucose AUC/g by age of horse GI (SEM) Treatment Dextrose Whole oats Rolled oats High fat rice bran Pelleted corn Cracked corn Pelleted steamprocessed corn EnergX [expanded 1] EnergX [expanded 2] EnergX [pelleted] EnergX [unprocessed]
Overall Mean d,e,f
Glucose AUC/g (SEM) 2-Year-Olds
Mature
Overall Mean a
2-Year-Olds
Mature
100 (19) 136 (19)c,d 208 (19)b 133 (19)c,d,e 213 (19)b 84 (19)e,f 278 (19)a
100 (25) 142 (26) 190 (26) 128 (26) 205 (25) 57 (25) 267 (26)
100 (31) 129 (29) 228 (29) 139 (29) 222 (31) 116 (31) 292 (29)
1.25 (0.10) 0.30 (0.10)c,d,e 0.49 (0.10)b,c,d 0.53 (0.10)b,c 0.47 (0.10)b,c,d 0.18 (0.10)e 0.72 (0.10)b
1.33 (0.13) 0.36 (0.14) 0.48 (0.14) 0.34 (0.14) 0.51 (0.13) 0.15 (0.13) 0.86 (0.14)
1.16 (0.14) 0.24 (0.13) 0.49 (0.13) 0.73 (0.13) 0.43 (0.14) 0.21 (0.14) 0.57 (0.13)
170 (19)b,c 144 (19)c,d 156 (19)c 81 (20)f
172 (26) 154 (25) 147 (26) 75 (25)
169 (29) 132 (31) 166 (29) 89 (33)
0.38 (0.10)c,d,e 0.34 (0.10)c,d,e 0.36 (0.10)c,d,e 0.32 (0.10)c,d,e
0.45 (0.14) 0.40 (0.13) 0.39 (0.14) 0.46 (0.13)
0.30 (0.13) 0.28 (0.14) 0.33 (0.13) 0.18 (0.15)
Overall mean treatment glycemic indices (GI) were calculated by taking the area under the curve (AUC) of the plasma glucose for each treatment divided by the AUC of the dextrose control and multiplying by 100. Parameters are relative to baseline measurements. a,b,c,d,e,f Means within a column not sharing a common superscript differ (P % .05).
Table 3. Overall mean treatment peak plasma glucose (GLU) concentrations (mg/dL), the treatment peak plasma GLU concentrations by age of horse, overall mean treatment time (minute) to peak plasma glucose concentrations, and the treatment time to peak plasma glucose concentrations by age of horse with the amount consumed used as a covariate for determination of peak GLU Peak GLU (SEM) Treatment Dextrose Whole oats Rolled oats High fat rice bran Pelleted corn Cracked corn Pelleted steamprocessed corn EnergX [expanded 1] EnergX [expanded 2] EnergX [pelleted] EnergX [unprocessed]
Overall Peak
2-Year-Olds
Time to Peak GLU (SEM) Mature*
b,c,d
141 (6) 123 (4)e,f,g 143 (4)b,c,d 118 (4)f,g 150 (4)b 115 (4)g 165 (4)a
143 (7) 126 (6) 147 (6) 122 (6) 158 (6) 113 (6) 172 (6)
139 (8) 120 (6) 139 (6) 120 (6) 143 (6) 117 (6) 157 (6)
134 (4)c,d,e 127 (4)d,e,f 116 (4)f,g 115 (4)g
138 (6) 134 (6) 116 (6) 116 (6)
130 (6) 119 (6) 117 (6) 116 (6)
Overall Time
2-Year-Olds
Mature*
76 (17) 104 (12)c,d 128 (12)a,b,c 106 (12)b,c,d 141 (12)a 89 (12)d 137 (12)a,b
99 (21) 121 (18) 122 (18) 124 (18) 147 (16) 91 (16) 117 (18)
53 (22) 87 (16) 134 (16) 87 (17) 135 (18) 88 (18) 158 (16)
101 (12)c,d 109 (12)a,b,c,d 137 (12)a,b 107 (12)a,b,c,d
114 (18) 118 (16) 144 (18) 101 (17)
87 (17) 100 (18) 129 (16) 113 (18)
d
Parameters are relative to baseline measurements. a,b,c,d,e,f,g Means not sharing a common superscript differ (P % .05). * Overall mature different than 2-year-old (P < .01).
The plasma glucose AUC/g starch consumed did not differ by age but there was a treatment difference (P < .01), with the high fat rice bran having a higher response than all other treatments (Table 6). In contrast, serum insulin AUC/g starch was greater for the mature horses than for the 2-year-olds (P < .01).
Treatment differences were detected (P < .05) with whole oats and high fat rice bran having a greater response than cracked corn. Within treatments, mature horses had a higher response than the 2-year-olds (P < .05) when consuming whole oats, pelleted corn, and EnergX [unprocessed].
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Table 4. Overall mean treatment insulinemic index (II), the treatment II by age of horse, overall mean serum insulin area under the curve (mIU/mL/min) divided by the grams of treatment consumed (insulin AUC/g), and the treatment insulin AUC/g by age of horse II (SEM) Treatment Dextrose Whole oats Rolled oats High fat rice bran Pelleted corn Cracked corn Pelleted steamprocessed corn EnergX [expanded 1] EnergX [expanded 2] EnergX [pelleted] EnergX [unprocessed]
Insulin AUC/g (SEM)
Overall Mean e
2-Year-Olds
Mature*
Overall Mean
2-Year-Olds
Mature*
a
100 (46) 217 (46)b 216 (46)b 107 (46)e 372 (46)a 111 (46)d,e 377 (46)a
100 (161) 178 (168) 294 (168) 201 (168) 294 (161) 69 (161) 373 (168)
100 (41) 227 (39)** 197 (39)** 85 (39) 391 (41)** 121 (41) 378 (39)**
1.28 (0.14) 0.53 (0.14)c 0.59 (0.14)c 0.37 (0.14)c 0.89 (0.14)b 0.30 (0.14)c 0.93 (0.14)b
0.63 (0.20) 0.21 (0.21) 0.32 (0.21) 0.23 (0.21) 0.32 (0.20) 0.09 (0.20) 0.42 (0.21)
1.93 (0.21)** 0.86 (0.20)** 0.85 (0.20) 0.51 (0.20) 1.47 (0.21)** 0.51 (0.21) 1.45 (0.20)**
206 (46)b,c,d 184 (46)b,c,d,e 208 (46)b,c 117 (46)c,d,e
277 (168) 243 (161) 161 (168) 73 (161)
189 (39)** 170 (41)** 219 (39)** 128 (41)**
0.51 (0.14)c 0.52 (0.14)c 0.51 (0.14)c 0.49 (0.14)c
0.31 (0.21) 0.28 (0.20) 0.19 (0.21) 0.13 (0.20)
0.71 (0.31) 0.28 (0.20) 0.83 (0.20)** 0.86 (0.21)**
Overall mean treatment insulinemic index (II) were calculated by taking the area under the curve (AUC) of the serum insulin for each treatment divided by the AUC of the dextrose control and multiplying by 100. Parameters are relative to baseline measurements. a,b,c,d,e Means within a column not sharing a common superscript differ (P % .05). * Overall mature different than 2-year-old (P < .01). ** Mature different than 2-year-old within a given treatment (P % .05).
Table 5. Overall mean treatment peak serum insulin (INS) concentrations (mIU/mL), the treatment peak serum INS concentrations by age of horse, overall mean treatment time (min) to peak serum insulin concentrations, and the treatment time to peak serum insulin concentrations by age of horse with the amount consumed used as a covariate for determination of peak INS Peak INS (SEM) Treatment Dextrose Whole oats Rolled oats High fat rice bran Pelleted corn Cracked corn Pelleted steamprocessed corn EnergX [expanded 1] EnergX [expanded 2] EnergX [pelleted] EnergX [unprocessed]
Overall Peak
Time to Peak INS (SEM) 2-Year-Olds
Mature*
b,c
84 (19) 86 (13)b,c 76 (13)b,c 61 (13)c 135 (13)a 51 (13)c 108 (13)a,b
51 (22) 26 (19) 39 (19) 30 (19) 39 (18) 19 (18) 49 (19)
118 (23) 146 (18) 114 (18) 93 (18) 231 (19) 83 (19) 168 (18)
79 (13)b,c 76 (13)b,c 68 (13)c 57 (13)c
31 (19) 33 (18) 22 (19) 31 (19)
128 (18) 119 (19) 115 (18) 82 (19)
Overall Time
2-Year-Olds
Mature*
88 (20) 115 (14)a,b,c 141 (14)a 110 (14)a,b,c 133 (14)a,b 94 (14)c 117 (14)a,b,c
109 (24) 116 (20) 139 (20) 82 (19) 135 (19) 119 (19) 132 (20)
67 (25) 114 (19) 144 (19) 82 (19) 132 (20) 69 (20) 103 (19)
94 (14)c 89 (14)c 140 (14)a 94 (14)b,c
113 (20) 106 (19) 167 (20) 109 (20)
75 (19) 72 (20) 112 (19) 80 (20)
c
Parameters are relative to baseline measurements. a,b,c Means within a column not sharing a common superscript differ (P % .05). * Overall mature different than 2-year-olds (P < .01).
DISCUSSION AND CONCLUSION When designing projects to evaluate glucose and insulin responses in horses, many factors must be considered. Unlike with human beings, no standardized glycemic or II has
been formulated for the equine,15 although Rodiek and Stull16 published a GI of some common equine feeds with the GI of oats being set equal to 100. As would be expected, feeds having higher starch content were found to
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Table 6. Overall mean treatment plasma glucose area under the curve (mg/dL*min) divided by the grams of starch consumed (glucose AUC/g starch), the treatment glucose AUC/g starch by age of horse, overall mean serum insulin area under the curve (mIU/mL*min) divided by the grams of starch consumed (insulin AUC/g starch), and the treatment insulin AUC/g starch by age of horse Glucose AUC/g Starch (SEM)
Insulin AUC/g Starch (SEM)
Treatment
Overall Mean
2-Year-Olds
Mature
Overall Mean
2-Year-Olds
Mature*
Dextrose Whole oats Rolled oats High fat rice bran Pelleted corn Cracked corn Pelleted steamprocessed corn EnergX [expanded 1] EnergX [expanded 2] EnergX [pelleted] EnergX [unprocessed]
NA 1.1 (0.3)b 0.9 (0.3)b 2.4 (0.3)a 0.7 (0.3)b 0.3 (0.3)b 1.1 (0.3)b
NA 1.3 (0.5) 0.9 (0.5) 1.5 (0.5) 0.8 (0.4) 0.3 (0.4) 1.4 (0.5)
NA 0.8 (0.4) 0.9 (0.4) 3.3 (0.4) 0.6 (0.5) 0.3 (0.5) 0.9 (0.4)
NA 1.9 (0.4)a 1.1 (0.4)a,b 1.6 (0.4)a 1.3 (0.4)a,b 0.5 (0.4)b 1.5 (0.4)a,b
NA 0.7 (0.6) 0.6 (0.6) 1.0 (0.6) 0.5 (0.6) 0.1 (0.6) 0.7 (0.6)
NA 3.1 (0.6)** 1.6 (0.6) 2.3 (0.6) 2.1 (0.6)** 0.9 (0.6) 2.2 (0.6)
0.9 (0.3)b 0.9 (0.3)b 0.8 (0.3)b 0.6 (0.3)b
1.0 (0.5) 1.1 (0.4) 0.8 (0.5) 1.2 (0.4)
0.7 (0.4) 0.8 (0.5) 0.7 (0.4) 0.0 (0.5)
1.2 (0.4)a,b 1.4 (0.4)a,b 1.1 (0.4)a,b 1.3 (0.4)a,b
0.7 (0.6) 0.7 (0.6) 0.4 (0.6) 0.3 (0.6)
1.6 (0.6) 2.1 (0.6) 1.8 (0.6) 2.4 (0.6)**
Parameters are relative to baseline measurements. a,b Means within a column not sharing a common superscript differ (P % .05). * Overall mature different than 2-year-old (P < .01). ** Mature different than 2-year-old within a given treatment (P % .05).
have the highest GI. Hoekstra et al used glucose AUC for cracked corn as the response to which other treatments were compared.6 Limitations to using feeds such as oats or corn as the reference are that they can vary both in quantity of starch as well as the availability of that starch. Additionally, rate of consumption of a reference feed can influence results.17 Using dextrose as a control helps to ensure that similar responses can be expected between studies; however, it can be administered both orally and injected intravenously.12 The oral dosage chosen (0.25 g dextrose/kg of BW) was between the 0.2 g dextrose/kg of BW oral dose used by Hoffman et al18 and the 0.3 g dextrose/kg of BW intravenous dose used by the same laboratory.19 This dosage of feed resulted in glycemic indices ranging from 81 to 278 in the current study when horses were fed their dietary treatments at the rate of 1.5 g/kg of BW. Another consideration in choosing the dosage of the dextrose control is that, as an oral drench, the dextrose is immediately consumed, whereas the other treatments are consumed over a period. Hence, even though the amount of dextrose being provided is slightly lower than the amount of starch that was provided in the feed with the lowest starch content (high fat rice bran), the glucose and insulin responses of many of the treatments were similar to the response of the dextrose control, suggesting the dosage may have been appropriate – especially given that only one treatment had peak glucose concentrations and only two treatments had peak insulin concentrations higher than the dextrose control.
The amount of test feedstuff to be administered is another variable that differs between studies. When fed on an equal available carbohydrate basis (2 g carbohydrate/kg of BW), a similar plasma glucose AUC was reported in Thoroughbreds given corn, oat groats, and rolled barley.20 The glucose and insulin peaks, as well as AUC responses, did not vary by processing method in Standardbreds fed 630 g starch per day from oats or corn at a moderate intake (between 1.2 and 1.5 g starch/kg of BW).21,22 By virtue of providing similar amounts of starch, it would reason that detecting differences between treatments would be difficult even with different availability of that starch. Another technique included feeding an amount of feed calculated to provide 4 Mcal digestible energy (DE) per meal.16 Although this technique resulted in differences in glycemic responses, the DE of a feedstuff was calculated from book values and simply represents averages that could vary greatly from the actual DE of the feedstuffs tested. Thus, in this study, it was decided the testing should be done on a standard weight of feed per kg of BW basis. Furthermore, most horse owners provide feed on the basis of the weight of the feedstuffs given, as opposed to starch content or energy content. Although those factors influence how much feed is given, it is the comparison of equal quantities of feeds that tend to be of the most practical benefit for horse owners. Of note, the amount of treatment being administered was based off the weight of each horse from the previous week, although the amount consumed was later calculated
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from the weight determined just before testing with young horses losing an average of 1.3 kg/wk and the mature horses losing an average of 2.2 kg/wk. This resulted in minor variations from the target intake of 1.5 g/kg of BW even when all the feedstuff was consumed. Although most horses consumed all of the treatments offered to them, the high fat rice bran and EnergX [unprocessed] had lower consumption rates. The EnergX [unprocessed] also had the lowest GI. Excluding that treatment, one could argue that the dextrose dosage could be increased given that whole oats, used as the reference in some other studies, had a GI of 136 and only one feed that was entirely consumed had a GI lower than the control. However, it should be pointed out that all of the feeds being tested were concentrates that would be expected to have a high GI – especially compared with forages or some by-products.16 Additionally, a dosage amount of between 0.20 and 0.25 g dextrose/kg of BW represents an amount of glucose somewhat equivalent to the amount often consumed in a grain meal.17 Although the horses did not receive any concentrate other than the test feedstuffs during the course of the study, horses had prior familiarization with eating rolled oats and cracked corn suggesting that a lack of familiarity with unprocessed EnergX and high fat rice bran may have been the reason for incomplete consumption. Glycemic response has been used as an indirect measure of prececal starch digestibility.6 Although attention has been paid to the glycemic response of various feeds to minimize insulin resistance,23 a low response could indicate starch is escaping to the hindgut of the horse - an undesirable consequence. With this in mind, two trials (data not shown) were conducted using ileal cannulated pigs at the Cargill Animal Nutrition Innovation Campus to determine prececal starch digestion of some of the same ingredients, processed identically, used in this trial. The pigs, averaging 41 kg, were fed on an equal starch basis at the rate of 1.9% starch/kg of BW. Pigs were used as a model for equine because of the similar small intestine anatomy and physiology, although a-amylase activity has been shown to be lower in horses than in pigs.7 When data from those trials were combined, the ileal starch digestibility of EnergX pellet (97.0%), EnergX unprocessed (96.2%), EnergX expanded 1 (95.8%), EnergX expanded 2 (94.9%), corn pellet (95.2%), and pelleted steam corn (96.9%) were similar. Other ingredients such as cracked corn (89.8%), whole oats (91.3%), and steam rolled oats (90.4%) had a slightly lower ileal starch digestibility. The data from the horse trial and the pig trial could explain the low GI of the cracked corn because it would rank as one of the lower starch digestibility ingredients used in this pig trial. However, when the corn was processed, the risk of starch leaking to the cecum would decrease because of an increased amount of starch digestibility by about 5%–6%. It also makes sense
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that lower starch ingredients that have equal starch digestibility of higher starch ingredients would have less starch available to leak to the cecum of equine. An example of this can be seen when comparing EnergX pellet versus pelleted steam-processed corn. Both of these ingredients have a starch digestibility of approximately 97% but total starch in the EnergX pellet was 45.6% and in pelleted steamprocessed corn was 64.1%, which leads to an approximate 27% reduction in the amount of starch available to leak into the hindgut. Although age did not have a detectable effect on GI or the glucose AUC/g of feed consumed, there were differences by treatment. Several treatments had a greater GI than did the control. Processing through rolling, pelleting, and expanding increased the GI of the tested feedstuffs, although the ‘‘expanded 2’’ method of processing designed to increase the gelatinization of starch produced only a trend for an increase over the control. This was probably because of the fact that the ‘‘expanded 2’’ product actually had a lower gelatinization of starch relative to the ‘‘expanded 1’’ product. Gelatinization is the irreversible swelling and destruction of the crystalline structure of the starch granules,24 which can result in the starch becoming more available for enzymatic digestion. Although physical processing such as grinding of grains affects digestibility of starch prececally, the effect is not as dramatic as thermal and hydrothermal processing techniques.7 Hoekstra et al reported the highest GI for steam-flaked corn, relative to cracked or ground, and suggested it reflects an increased prececal starch digestibility because of thermal processing.6 This conclusion regarding prececal improvement in starch digestibility of grains because of thermal processing is supported in the pig ileal starch digestibility data presented earlier. In contrast to the processed grains, all unprocessed grains, cracked corn, and the high fat rice bran did not differ from the control in GI, suggesting that the total starch digested in these treatments was lower (as was the case with high fat rice bran which contained only 22.5% starch) or less available to digestion in the foregut. For example, it has been reported that whole corn had lower prececal starch digestibility than oats and also resulted in lower blood glucose concentrations when fed to ponies.25 High fat rice bran had the second highest AUC when standardized by accounting for differences in intake, thus suggesting that the lower GI was due to a reduced consumption rate. By comparison, although horses consumed less EnergX [unprocessed] than the other treatments (excluding the control), when standardized by evaluating the AUC on the basis of amount eaten, it did not differ from the processed EnergX treatments. Although the rate of intake was shown to not correlate with the AUC for blood glucose concentration in a study by Harris et al,26 differences in the amount consumed certainly
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would affect results and need to be considered. The pelleted steam-processed corn continued to elicit the greatest response (excluding the control), whereas the cracked corn produced the lowest when based on the amount consumed. Although peak plasma glucose concentrations were greater with the 2-year-old horses, the difference was not dramatic (roughly 5% difference). The greater peak plasma glucose concentration reported in the pelleted corn and pelleted steam-processed corn treatments presumably reflects a greater availability of starch for prececal digestion. Interestingly, Vervuert et al reported no association of the highest degree of gelatinization in different barley types with the most pronounced glycemic or insulinemic responses.27 The time until peak glucose concentrations was reached was longer with the 2-year-olds than mature horses, which may simply be a reflection of the consumption rate. Pelleted corn and pelleted steam-processed corn exhibited a longer time until peak glucose concentrations, potentially because of the large amount of starch available for digestion. Besides consumption rate, as suggested in this study, Pagan et al demonstrated increasing intake from 0.75 kg up to 1.5 kg increased time to peak glucose concentration.28 Relatively few studies in horses have examined the relationship between glycemic and insulinemic responses15 despite the attention paid to the glycemic response of horses to various feeds to try and control insulin resistance.23 A striking revelation of this study is the difference in insulin response between mature and 2-year-old horses. The mature horses exhibited a much higher insulin response despite having relatively similar glucose concentrations, suggesting that the mature horses had reduced insulin sensitivity. Both the pelleted corn and the pelleted steam-processed corn yielded the greatest insulin response to a meal, as represented by the II, which reflects the high glucose response of both treatments. Whole oats and rolled oats produced the next highest II, which did not differ from the II for the three processed EnergX treatments. As with the plasma glucose concentrations, the high fat rice bran, cracked corn, and EnergX [unprocessed] produced the lowest response, which did not differ from the control. Additionally, the EnergX [expanded 2] treatment did not differ from the control, suggesting the increased starch gelatinization desired in the processing steps did not occur. When the serum insulin AUC was divided by the amount of treatment consumed, the dextrose treatment yielded the highest results. Pelleted corn and pelleted steamprocessed corn were intermediary and the other treatments were the lowest, suggesting a decrease in insulin release with these other treatments. Of note, when the serum insulin AUC was divided by the amount of starch consumed, cracked corn yielded the lowest result, suggesting, once again, a lower digestibility of the starch.
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The peak insulin concentrations were about 4 times greater in the mature horses (127 mIU/mL) than in 2-year-olds (33.6 mIU/mL), despite 2-year-old horses having higher peak plasma glucose concentrations. The treatment differences seen with peak serum insulin concentrations reflect the variations seen in peak plasma glucose concentrations, as do the differences in time to peak serum insulin concentrations with the peak occurring later in the 2-year-olds. This, too, likely reflects the slower feed consumption rate by the 2-year-olds. The elevated insulin response in the mature Arabian horses is an intriguing finding with implications for horse health. It has been suggested that horses on a low-starch diet were less efficient in metabolizing blood glucose than those consuming a grain-based diet.29 Higher glycemic responses would be expected in horses unaccustomed to receiving high GI feeds (such as those on this study) as compared to horses that are adapted to them.18 Additionally, horses consuming only forages would be expected to have decreased rates of glucose clearance compared with ones that are fed high starch concentrates when subjected to a standardized dextrose challenge. Horses may adapt to a diet rich in starch and sugar and the result may be a moderated glucose response. However, insulin sensitivity has also been shown to decrease when horses were fed a diet rich in starch and sugar.19 This can be of great concern when managing obese horses as they can have an increased likelihood of insulin resistance.19 Using the Minimal Model approach to access glucose and insulin dynamics, Treiber et al reported lower insulin sensitivity in ponies with a history of recurrent laminitis on spring pasture.9 In this case, the insulin sensitivity was not related to obesity although ponies have been shown to have a higher degree of insulin resistance than Dutch Warmblood horses.30 In the present study, the mean body condition score did not differ between age groups (2-year-olds ¼ 5.6; mature ¼ 6.4) and did not clearly correlate to the responses of the horses. Although it is important to minimize starch to the equine large intestine by having high prececal starch digestibility, high glucose and insulin responses have been associated with health issues in human beings.31 The same concern likely exists with horses and a controlled response—one in which the glucose and insulin concentrations in the blood are not exaggerated but little starch escapes to the hindgut – potentially may be the ideal goal for which to strive. In populations where insulin insensitivity is an issue, such as in this population of mature Arabians, proper selection of dietary constituents, including the method in which feedstuffs are processed, may aid in preventing various health problems. These data indicate that an insightful awareness of the mode of glycemic response is needed to deliver a diet that will successfully manage the response. A low glycemic
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response is not always best as can be seen in the cracked corn data and the potential for cecal starch leakage to occur. A low glycemic response can deliver a desired response if the starch is nearly completely digested prececally. Also, thermal processing of ingredients can have a major effect on glycemic and insulinemic responses in horses. Although starch and sugar concentrations are an important part of controlling glycemic and insulinemic responses, other nutrients such as gelatinized starch, fat, and fiber play in important role.
14. Wolver TMS, Jenkins DJA, Jenkins AL, Josse RG. The glycemic index: methodology and clinical implications. Am J Clin Nutr 1991; 54:846–854. 15. Vervuert I, Coenen M. Glycaemic index of feeds for horses. Pferdeheilkunde 2005;21:79–82. 16. Rodiek AV, Stull CL. Glycemic index of ten common horse feeds. J Equine Vet Sci 2007;27:205–211. 17. Ralston SL. Factors affecting glucose and insulin metabolism in young horses. Pferdehilkunde 2005;21:83–86. 18. Hoffman RM, Kronfeld DS, Cooper WL, Harris PA. Glucose clearance in grazing mares is affected by diet, pregnancy, and lactation.
ACKNOWLEDGMENTS Funding for this project was provided by Cargill Animal Nutrition, Minnetonka, MN. REFERENCES 1. Ralston SL. Glucose intolerance and developmental orthopedic disease in foals—a connection? Proc Equine Nutr Conf Feed Manufacturers 1999;151–156. 2. Pagan JD, Geor RJ, Caddel SE, Pryor PB, Hoekstra KE. The
J Anim Sci 2003;81:1764–1771. 19. Hoffman RM, Boston RC, Stefanovski D, Kronfeld DS, Harris PA. Obesity and diet affect glucose dynamics and insulin sensitivity in thoroughbred geldings. J Anim Sci 2003;81:2333–2342. 20. Jose-Cunilleras E, Taylor LE, Hinchcliff KW. Glycemic index of cracked corn, oat groats and rolled barley in horses. J Anim Sci 2004;82:2623–2629. 21. Vervuert I, Coenen M, Bothe C. Effects of oat processing on the glycaemic and insulin responses in horses. J Anim Physiol Anim Nutr 2003;87:96–104.
relationship between glycemic response and the incidence of OCD
22. Vervuert I, Coenen M, Bothe C. Effects of corn processing on the
in thoroughbred weanlings: a field study. Proc Am Assoc Equine
glycaemic and insulinaemic responses in horses. J Anim Physiol Anim Nutr 2004;88:348–355.
Pract 2001;47:322–325. 3. Treiber KH, Boston RC, Kronfeld DS, Staniar WB, Harris PA. Insu-
23. Kronfeld DS, Treiber KH, Hess TM, Boston RC. Insulin resistance
lin resistance and compensation in thoroughbred weanlings adapted
in the horse: definition, detection, and dietetics. J Anim Sci 2005;
to high-glycemic meals. J Anim Sci 2005;83:2357–2364. 4. Treiber KH, Kronfeld DS, Geor RJ. Insulin resistance in equids:
24. Selmi B, Marion D, Perrier Cornet JM, Douzala JP, Gervais P.
possible role in laminitis. J Nutr 2006;139:2094S–2098S.
83:1–10. Amyloglucosidase hydrolysis of high-pressure and thermally gelati-
5. Stull CL, Rodiek AV. Responses of blood glucose, insulin and cortisol
nized corn and wheat starches. J Agric Food Chem 2000;48:2629–
concentrations to common equine diets. J Nutr 1988;118:206–213. 6. Hoekstra KE, Newman K, Kennedy MAP, Pagan JD. Effect of corn
2633. ¨ ber den einfluß von futterart und 25. Radicke S, Meyer H, Kienzle E. U
processing on glycemic response in horses. Proc Equine Nutr Conf
fu¨tterungszeitpunkt auf den blutglucosespiegel bei pferden. Pferde-
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7. Julliand V, De Fombelle A, Varloud M. Starch digestion in horses: the impact of feed processing. Livestock Sci 2006;100:44–52.
26. Harris PA, Sillence M, Inglis R, Siever-Kelly C, Friend M, Munn K,
8. Jose-Cunilleras E, Hinchcliff KW, Sams RA, Devor ST, Linderman JK.
take rate and glycaemic response of sweet feed. Pferdeheilkunde
Glycemic index of a meal fed before exercise alters substrate use and glucose flux in exercising horses. J Appl Physiol 2002;92:117–128. 9. Treiber KJ, Hess TM, Kronfeld DS, Boston RC, Geor R, Harris PA. Insulin resistance and compensation in laminitis-predisposed
Davidson H. Effect of short (<2 cm) lucerne chaff addition on the in2005;21:87–88. 27. Vervuert I, Bother C, Coenen M. Glycaemic and insulinaemic responses to mechanical or thermal processed barley in horses. J Anim Physiol Anim Nutr 2007;91:263–268.
ponies characterized by the Minimal Model. Pferdeheilkunde
28. Pagan JD, Harris PA, Kennedy MAP, Davidson N, Hoekstra KE.
2005;21:91–92. 10. Murphy D, Reid SWJ, Love S. The effect of age and diet on the oral
Feed type and intake affect glycemic response in thoroughbred
glucose tolerance test in ponies. Equine Vet J 1997;29:467–470.
horses. Proc Equine Nutr Conf Feed Manufacturers 1999;147–149. 29. Ott EA, Brown MP, Roberts GD, Kivipelto J. Influence of starch in-
11. Gordon ME, Jerina ML, Raub RH, Davison KE, Young JK.
take on growth and skeletal development of weanling horses. J Anim
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Sci 2005;83:1033–1043. 30. Rihnen KEPM, van der Kolk JH. Determination of reference range
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values indicative of glucose metabolism and insulin resistance by
12. Ralston SL. Insulin and glucose regulation. Vet Clin Equine 2002;
use of glucose clamp techniques in horses and ponies. Am J Vet
18:295–304. 13. Xiong Y, Bartel SJ, Preston RL. Improved enzymatic method to
31. Vervuert I, Coenen M. The glycemic and insulinemic index in horses.
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ABSTRACT The objective of this study was to examine age-related differences in glycemic and insulinemic responses of horses that were fed various feedstuffs, with particular attention to method of feed processing. A 16 16 Latin square design was used with eight 2-year-olds and eight mature Arabians. Horses were maintained on a roughage diet and were subjected to a glycemic response test once weekly. A control treatment consisted of an oral dextrose drench (0.25 g dextrose/kg of BW). Ten treatments consisted of variously processed feed ingredients fed at the rate of 1.5 g/kg of BW. Five other treatments were commercial feeds of a proprietary nature and are not reported. Fasting blood samples were taken once a week for 16 weeks. Thirty minutes later, another baseline sample was taken and horses were administered their respective treatment. Further blood samples were taken every 30 minutes through four hours. Samples were analyzed for glucose and insulin concentrations. Differences in glucose response between 2-year-olds and mature horses were minimal. However, mature horses had a higher insulin response (P < .01) suggesting young horses had greater insulin sensitivity. Additionally, differences (P < .05) existed between treatments with pelleted steam-processed corn having the highest glycemic response and cracked corn the lowest. Results from this study confirm that mature horses have reduced insulin sensitivity and that both glycemic and insulinemic responses are altered with feed processing techniques. Thermal processing produces the greatest response; however, a low glycemic response may not be desirable if starch escapes into the hindgut. Keywords: Feed processing; response; Horse; Insulin
Glucose;
Glycemic
From theDepartment of Animal Science, Michigan State University, East Lansing, MIa; Davis College of Agriculture, Forestry and Consumer Sciences, West Virginia University, Morgantown, WVb; and Cargill Animal Nutrition, Minnetonka, MNc. Reprint requests: Brian D. Nielsen, PhD, Department of Animal Science, Michigan State University, 1287D Anthony Hall, East Lansing, MI 48824. 0737-0806/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2010.03.008
Journal of Equine Veterinary Science Vol 30, No 5 (2010)
Many research studies have been conducted to investigate the glycemic responses of horses to various feedstuffs. The demand for this research has been driven, in part, by the theory that insulin resistance increases the risk of certain diseases, such as laminitis and osteochondrosis,1-4 and such insulin resistance may be affected by the glycemic characteristics of the diet.1 Previous studies have demonstrated that feed type, intake, and feed processing all contribute to varying glycemic responses in horses.5-7 Additionally, the glycemic index (GI) of a meal has been shown to alter glucose use during exercise in horses.8 Although the glycemic and insulinemic responses of various feeds have been examined in weanlings,2,9 the comparison of responses in young horses with that of mature horses is limited. Murphy et al compared the plasma glucose response to an oral glucose tolerance test in both weanling and mature ponies, with lower plasma glucose concentrations being reported in mature ponies.10 However, insulin was not measured. When an identically formulated concentrate was tested in three forms (5-mm extruded, 4-mm pellet, 19-mm pellet), there were minimal differences between glucose and insulin responses, but the concentrate was formulated to be low in sugar and starch.11 Whether the same would be true for more traditional feedstuffs is unclear. Therefore, the hypothesis of this study is that young horses have a lower insulin response than the mature horses when fed various feedstuffs.
MATERIALS AND METHODS Animal Care and Management A 16 16 Latin square design was used to test the response of 15 variously processed feedstuffs and commercial feeds designed for horses, along with an oral dextrose drench that was used as a control. Sixteen Arabian horses, specifically four 2-year-old geldings (414 3 kg; 28 1 month), four 2-year-old fillies (397 3 kg; 28 1 month), four mature geldings (480 2 kg; 14.3 0.5 year), and four mature mares (466 2 kg; 14.1 0.5 year), were maintained on pasture with access to a trace-mineralized salt block. Horses received light exercise either through horsemanship classes (mature horses) or through training classes (2-year-old horses) for approximately 1 hour 3–4 days per
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week for the duration of the study. Horses were supplemented with grass hay but received no concentrate to ensure they were on what would be considered a low glycemic-response diet. Each horse was tested for glycemic and insulinemic response once per week for 16 weeks, with testing done twice a week on eight horses per day. The afternoon before each test, the horses were brought into stalls from their pasture, weighed on a digital scale, and fed 0.5 % BW in timothy hay. All hay provided was consumed by the next morning with no refusals. Horses were kept in their individual stalls until sampling was completed. Sampling was conducted the following morning after a 12-hour fast to normalize blood glucose and insulin concentrations.12 Horses had ad libitum access to water throughout the study. The morning of sampling, each horse was catheterized with a 14-cm, 14-G catheter placed into either the left or the right jugular vein. An extension set with a stopcock was placed onto the catheter and sutured into place. The catheters were in place 30 minutes before the first blood sample, which was typically taken between 07:00 and 08:00 AM. After the final blood draw of each test, the catheters were removed and horses were returned to the pasture. The protocol was approved by the Michigan State University Institutional Animal Care and Use Committee (AUF# 09/06-117-0). Treatments A control treatment consisted of dextrose administered at the dosage of 0.25 g dextrose/kg of BW in a 50% oral drench (with water). Ten treatments consisted of variously processed ingredients used in equine feeds and included whole oats, rolled oats, high fat rice bran, pelleted corn, cracked corn, pelleted steam-processed corn, EnergX [expanded 1], EnergX [expanded 2], EnergX [pelleted], and EnergX [unprocessed], where EnergX is a unique combination of food grade corn particles from the Cargill Dry Corn Ingredient business unit (Paris, IL, USA). The grain used to make EnergX is the result of an identity-preserved system, which starts with the selection of toxin-resistant hybrids that are contract grown and traced through the processing system to deliver a unique, consistent nutrient profile for equine feeds. The difference between the two expanded types of EnergX is that the ‘‘expanded 2’’ process involves an increase in the amount of heat and water used to increase the gelatinization of starch. Although the first type of expanding involves using about 5% steam (added in the conditioner and some on the barrel) and an internal dye head temperature of about 1438C, the second type of expanding involves using about 7.5% steam (again, added in the conditioner and some on the barrel) and an internal dye head temperature of about 1548C. To reduce variation in the corn treatments, the cracked, pelleted, and pelleted steam-processed corn were all made from the same corn source. The five other treatments consisted of various
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commercial feeds. Although used in the 16 16 Latin square design to evaluate potential overall differences in glucose or insulin responses associated with age or gender, the feeds were of a proprietary nature and were composed of various combinations of ingredients as opposed to single ingredients in the other 10 treatments. Hence, the individual means from those five proprietary feeds are not reported. Each of the 16 horses was tested using the control or one of the feedstuffs or feeds each week for 16 weeks in a balanced Latin square design where treatment order was varied from horse to horse to eliminate any potential carryover effects. The feeds or feedstuffs were administered at the rate of 1.5 g/kg of BW on an as-fed basis of their weight from the previous week. The amount of feed consumed within 1 hour of offering was recorded along with the time taken until feed consumption ceased. Any feed not consumed was subtracted from the amount fed to determine the total amount eaten. Sample Handling Feed was processed at Cargill Animal Nutrition Innovation Campus (Elk River, MN, USA) with the expanded products processed at Cargill Dry Corn Ingredients. The feeds were bagged with an identifying code to which the researchers were blinded and then shipped to the Michigan State University Horse Teaching and Research Center. Feed samples were retained for full nutrient analysis by Cargill Animal Nutrition. On arrival at Michigan State University, each feed was sampled for later starch analysis. Samples were stored at 208C until analyzed. Blood samples were taken at 30, 0 (just before administering the appropriate treatment), 30, 60, 90, 120, 150, 180, and 240 minutes after treatment. After each blood sample, the catheter was flushed with 10 mL of heparinized saline (10 U heparin/mL saline). The blood was then injected into vacutainer tubes with potassium oxalate and sodium fluoride for the plasma samples to be used for glucose determination and with no preservative for the serum samples to be used for insulin determination. Tubes were placed on ice until they could be centrifuged at 3,000 g for 10 minutes (plasma tubes) or 3,000 g for 25 minutes (serum tubes). Plasma and serum were stored at 208C until analyzed. Sample Analyses Cargill Animal Nutrition provided nutrient analyses of the various treatments except for total starch (Table 1). Samples were analyzed for the determination of dry matter (DM), crude protein (CP), acid detergent fiber (ADF), neutral detergent fiber (NDF), ether extract (EE), and gelatinized starch (CP, AOAC 968.06; ADF and NDF, AOAC 2002.04/973.18, EE, AOAC 920.39; ash, AOAC 942.05). Gelatinized starch was measured by the methods described by Xiong et al.13 The starch content
1.50 (0.04) 1.48 (0.11) 1.40 (0.21) 0.87 (0.65) 1.51 (0.05)a 1.44 (0.18)a,b 1.46 (0.21)a,b 1.04 (0.61)c
1.53 (0.05) 1.39 (0.24) 1.52 (0.03) 1.23 (0.54)
of the feedstuffs was analyzed at Michigan State University by digesting 0.5 g of sample in duplicate. In brief, samples were digested with 20 mL H2O, 10 mL acetate buffer, 0.8 mL HCl, and 250 mL of Crystalzyme 40 L (Valley Research, South Bend, IN) in a 558C water bath for 16 hours. After the digestion, the liquid was diluted to 200 mL, centrifuged, and then was analyzed for free glucose. Glucose was analyzed using a commercially available kit (Autokit Glucose, Wako Diagnostics, Richmond, VA) that was adapted to 96-well plates. In brief, 9 mL of standard, sample, or control were pipetted onto a 96-well plate. Then, 350 mL of buffer solution was added to each well and incubated at 368C for 18 minutes. The plate was then read on a UV spectrophotometer (SpectraMax 340, Molecular Devices, Sunnyvale, CA) at 505 nm. Insulin was analyzed using a commercially available RIA (Coat-A Count Insulin, Diagnostic Products Corp., Los Angeles, CA) as directed by the package insert. Duplicates were accepted when variation was at or below 5% for glucose and 10% for insulin.
Means of the amount eaten (all horses combined) not sharing a common superscript differ (P < .05). a,b,c,d
8.3 9.6 9.5 10.2 2.98 2.99 3.05 3.14 87.7 90.0 88.8 88.8
12.4 12.7 12.4 13.0
4.38 4.54 3.91 4.17
17.63 18.49 15.86 16.87
43.3 37.3 45.6 37.5
81.1 60.0 31.6 23.4
0.28 (0.06) 1.49 (0.06) 1.50 (0.03) 1.52 (0.02) 1.48 (0.09) 1.26 (0.43) 1.32 (0.50) 5.5 5.9 18.6 2.8 2.9 3.8 3.24 2.15 11.44 1.34 1.17 1.36 89.5 87.3 93.1 86.5 85.7 88.2
15.1 13.2 14.4 8.7 8.5 8.8
12.14 3.24 5.69 2.07 1.79 1.63
27.37 10.17 13.44 8.06 7.07 7.51
28.1 53.2 22.5 67.4 63.5 64.1
26.2 30.7 57.3 42.0 18.7 54.8
0.27 (0.05)d 1.42 (0.35)a,b 1.45 (0.17)a,b 1.29 (0.47)b 1.50 (0.07)a 1.38 (0.33)a,b 1.43 (0.35)a,b
0.27 (0.04) 1.36 (0.48) 1.41 (0.23) 1.09 (0.59) 1.53 (0.03) 1.52 (0.02) 1.53 (0.04)
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Dextrose Whole oats Rolled oats High fat rice bran Pelleted corn Cracked corn Pelleted steamprocessed corn EnergX [expanded 1] EnergX [expanded 2] EnergX [pelleted] EnergX [unprocessed]
Treatment
% Total All Horses 2-Year-Olds Mature Starch, Grams Eaten/ Grams Eaten/ Grams Eaten/ % DM % Ash % CP % Fat % ADF % NDF % Starch Gelatinized kg BW (SEM) kg BW (SEM) kg BW (SEM)
Table 1. Nutrient analyses of the feed ingredients tested and the average amount of each treatment consumed (g/kg BW SEM) on an as-fed basis
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Glycemic and Insulinemic Response Calculations The glycemic and insulin indices were determined by averaging the responses at 30 and 0 minutes pre-feeding for a baseline and then using the trapezoidal method of numerical integration14 to calculate the area under the curve (AUC) above baseline values. Statistical analyses were run on the AUC but to provide comparative indices to the control, the AUC for dextrose was set at 100 and the other treatments were reported relative to that, to determine both a GI and insulinemic index (II). To account for potential differences in the amount consumed, the AUC/g of feed and AUC/g starch eaten were also calculated. The peak plasma glucose and serum insulin concentrations above baseline were identified from the sample points, as were the times until the peak concentrations of both glucose and insulin were reached. Statistics Statistics on the 16 16 Latin square design were performed using SAS 8.2 (SAS Inst., Inc., Cary, NC, USA). PROC MIXED was used to evaluate glucose AUC, glucose AUC divided by grams of treatment consumed, insulin AUC, insulin AUC divided by grams of treatment consumed, and both the glucose and insulin AUC divided by grams of treatment and starch consumed, with day and horse nested in age as the random variables. PROC GLM was used to detect differences in peak glucose concentrations, time to peak glucose concentrations, peak insulin concentrations, and time to peak insulin concentrations. There was no affect of gender, and so it was removed from the model. The dependent variables were age, treatment, and age by treatment. Age was tested by horse nested in age. Treatment by age was tested by the residual. When main effect differences were detected, mean
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separation tests were performed. When the amount eaten on a kg/BW basis was found to be significant for an effect on peak plasma glucose and serum insulin concentrations, it was used as a covariate in the model.
RESULTS The amount of each treatment consumed on a g/kg of BW basis differed (P < .001; Table 1). By design, horses consumed less dextrose than the other tested treatments. Horses consumed less of the EnergX [unprocessed] than they did of the remaining treatments (P % .05). Horses also consumed more pelleted corn and EnergX [expanded 1] than they did of high fat rice bran (P % .05). Mature horses consumed the allotted feed faster (15.9 3.7 minutes) than did the 2-yearolds (33.7 3.9 minutes; P < .001). The GI as determined by the AUC did not differ by age (P ¼ .38) or age by treatment interaction (P ¼ .96). However, the GI of feeds differed (P < .001; Table 2) with rolled oats (GI ¼ 208), pelleted corn (GI ¼ 213), pelleted steamprocessed corn (GI ¼ 278), EnergX [expanded 1] (GI ¼ 170), and EnergX [pelleted] (GI ¼ 156) having a greater GI than the dextrose control (GI ¼ 100; P < .05). There was a trend for EnergX [expanded 2] (GI ¼ 144) to have a greater GI than the control (P ¼ .09). When the AUC for plasma glucose was evaluated on the basis of amount of feed consumed to account for differences in intake, there was no difference in the GI response by age (P ¼ .58) or age by treatment interaction (P ¼ .15). However, there was a treatment difference (P < .001; Table 2). The glucose AUC response on the basis of the amount eaten was greater with dextrose (1.25 mg/dL/min) than the other feed ingredients, which ranged from a high of 0.72 mg/dL/min for pelleted steam-processed corn to a low of 0.18 mg/ dL/min for cracked corn. Peak plasma glucose concentrations differed by age (P ¼ .002) and treatment (P < .001; Table 3). Two-year-old horses had a greater peak plasma glucose concentration (131.4 1.5 mg/dL) than did the mature horses (124.8 1.5 mg/dL). The peak plasma glucose concentration of pelleted steam-processed corn was higher than all other treatments. The lowest peak plasma glucose concentrations were recorded with the whole oats, high fat rice bran, cracked corn, EnergX [pelleted], and EnergX [unprocessed] treatments. Other significant variables in the model included the amount eaten on a kg BW basis, horse(age) and day, all at P < .01. Time to peak plasma glucose concentration was longer overall in 2-year-olds (P < .05) and varied by treatment (P < .05; Table 3). The 2-year-olds reached their peak plasma glucose concentrations at 124 4 minutes, whereas mature horses reached at 112 4 minutes. The shortest time to peak plasma glucose concentration occurred with the oral
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drench of dextrose (76 minutes), whereas the longest occurred with pelleted corn (141 minutes). There were age (mature horses greater than 2-year-olds; P ¼ .002), treatment (P < .001), and age by treatment interaction differences (P ¼ .001) for the average II (Table 4). High fat rice bran (II ¼ 107), cracked corn (II ¼ 111), EnergX [unprocessed] (II ¼ 117), and EnergX [expanded 2] (II ¼ 184) did not differ from the dextrose control (II ¼ 100), but both pelleted corn (II ¼ 372) and pelleted steam processed corn (II ¼ 377) were greater than all other treatments (P < .05). Although serum insulin AUC was greater for the mature horses than for the 2-year-olds (P ¼ .002), the age response is more apparent by examining the AUC for serum insulin (mIU/mL/min) on the basis of grams of treatment consumed (Table 4), in which there were also age (P ¼ .006), treatment (P < .001), and age by treatment interaction differences (P ¼ .04). The mature horses had an average insulin AUC/g consumed of 0.89 0.14 mIU/mL/min/g feed, whereas it was only 0.26 0.14 mIU/mL/min/g feed for the 2-year-olds. The dextrose treatment had the highest AUC/g consumed for insulin, whereas whole oats, rolled oats, high fat rice bran, cracked corn, and the four EnergX treatments had the lowest. For individual treatments, mature horses had greater serum insulin AUC/g consumed for dextrose, whole oats, pelleted corn, pelleted steam-processed corn, EnergX [pelleted], and EnergX [unprocessed]. Differences in peak serum insulin concentrations were detected for age and treatment (both at P < .001) and there was a trend for an age by treatment interaction difference (P ¼ .09; Table 5). Also having an effect on peak serum insulin concentrations were amount eaten per kg of BW (P ¼ .03) and horse(age) (P < .001) with a trend for a day difference (P ¼ .08). The overall peak serum insulin concentration was greater with mature horses (122 4 mIU/mL) than with the 2-year-olds (31 4 mIU/mL; P < .001). Pelleted corn had the greatest peak serum insulin concentration at 135 mIU/mL, with the pelleted steam processed corn being the next highest at 108 mIU/mL. Lowest peak serum insulin concentrations were with the cracked corn (51 mIU/mL), high fat rice bran (61 mIU/ mL), EnergX [unprocessed] (57 mIU/mL), and EnergX [pelleted] (68 mIU/mL). Differences in time to peak insulin concentrations existed for both age (P ¼ .001) and treatment (P ¼ .005; Table 5). As with peak glucose concentrations, peak serum insulin concentrations occurred at a later time with 2-year-olds (123 5 minutes) than with the mature horses (100 5 minutes; P ¼ .001). The shortest time was with the dextrose control (88 minutes), cracked corn (94 minutes), EnergX [expanded 1] (94 minutes), and EnergX [expanded 2] 89 minutes). The longest time was with rolled oats (141 minutes) and EnergX [pelleted] (140 minutes).
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Table 2. Overall mean treatment glycemic indices (GI), the treatment GI by age of horse, overall mean treatment plasma glucose area under the curve (mg/dL/min) divided by the grams of treatment consumed (glucose AUC/g), and the treatment glucose AUC/g by age of horse GI (SEM) Treatment Dextrose Whole oats Rolled oats High fat rice bran Pelleted corn Cracked corn Pelleted steamprocessed corn EnergX [expanded 1] EnergX [expanded 2] EnergX [pelleted] EnergX [unprocessed]
Overall Mean d,e,f
Glucose AUC/g (SEM) 2-Year-Olds
Mature
Overall Mean a
2-Year-Olds
Mature
100 (19) 136 (19)c,d 208 (19)b 133 (19)c,d,e 213 (19)b 84 (19)e,f 278 (19)a
100 (25) 142 (26) 190 (26) 128 (26) 205 (25) 57 (25) 267 (26)
100 (31) 129 (29) 228 (29) 139 (29) 222 (31) 116 (31) 292 (29)
1.25 (0.10) 0.30 (0.10)c,d,e 0.49 (0.10)b,c,d 0.53 (0.10)b,c 0.47 (0.10)b,c,d 0.18 (0.10)e 0.72 (0.10)b
1.33 (0.13) 0.36 (0.14) 0.48 (0.14) 0.34 (0.14) 0.51 (0.13) 0.15 (0.13) 0.86 (0.14)
1.16 (0.14) 0.24 (0.13) 0.49 (0.13) 0.73 (0.13) 0.43 (0.14) 0.21 (0.14) 0.57 (0.13)
170 (19)b,c 144 (19)c,d 156 (19)c 81 (20)f
172 (26) 154 (25) 147 (26) 75 (25)
169 (29) 132 (31) 166 (29) 89 (33)
0.38 (0.10)c,d,e 0.34 (0.10)c,d,e 0.36 (0.10)c,d,e 0.32 (0.10)c,d,e
0.45 (0.14) 0.40 (0.13) 0.39 (0.14) 0.46 (0.13)
0.30 (0.13) 0.28 (0.14) 0.33 (0.13) 0.18 (0.15)
Overall mean treatment glycemic indices (GI) were calculated by taking the area under the curve (AUC) of the plasma glucose for each treatment divided by the AUC of the dextrose control and multiplying by 100. Parameters are relative to baseline measurements. a,b,c,d,e,f Means within a column not sharing a common superscript differ (P % .05).
Table 3. Overall mean treatment peak plasma glucose (GLU) concentrations (mg/dL), the treatment peak plasma GLU concentrations by age of horse, overall mean treatment time (minute) to peak plasma glucose concentrations, and the treatment time to peak plasma glucose concentrations by age of horse with the amount consumed used as a covariate for determination of peak GLU Peak GLU (SEM) Treatment Dextrose Whole oats Rolled oats High fat rice bran Pelleted corn Cracked corn Pelleted steamprocessed corn EnergX [expanded 1] EnergX [expanded 2] EnergX [pelleted] EnergX [unprocessed]
Overall Peak
2-Year-Olds
Time to Peak GLU (SEM) Mature*
b,c,d
141 (6) 123 (4)e,f,g 143 (4)b,c,d 118 (4)f,g 150 (4)b 115 (4)g 165 (4)a
143 (7) 126 (6) 147 (6) 122 (6) 158 (6) 113 (6) 172 (6)
139 (8) 120 (6) 139 (6) 120 (6) 143 (6) 117 (6) 157 (6)
134 (4)c,d,e 127 (4)d,e,f 116 (4)f,g 115 (4)g
138 (6) 134 (6) 116 (6) 116 (6)
130 (6) 119 (6) 117 (6) 116 (6)
Overall Time
2-Year-Olds
Mature*
76 (17) 104 (12)c,d 128 (12)a,b,c 106 (12)b,c,d 141 (12)a 89 (12)d 137 (12)a,b
99 (21) 121 (18) 122 (18) 124 (18) 147 (16) 91 (16) 117 (18)
53 (22) 87 (16) 134 (16) 87 (17) 135 (18) 88 (18) 158 (16)
101 (12)c,d 109 (12)a,b,c,d 137 (12)a,b 107 (12)a,b,c,d
114 (18) 118 (16) 144 (18) 101 (17)
87 (17) 100 (18) 129 (16) 113 (18)
d
Parameters are relative to baseline measurements. a,b,c,d,e,f,g Means not sharing a common superscript differ (P % .05). * Overall mature different than 2-year-old (P < .01).
The plasma glucose AUC/g starch consumed did not differ by age but there was a treatment difference (P < .01), with the high fat rice bran having a higher response than all other treatments (Table 6). In contrast, serum insulin AUC/g starch was greater for the mature horses than for the 2-year-olds (P < .01).
Treatment differences were detected (P < .05) with whole oats and high fat rice bran having a greater response than cracked corn. Within treatments, mature horses had a higher response than the 2-year-olds (P < .05) when consuming whole oats, pelleted corn, and EnergX [unprocessed].
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Table 4. Overall mean treatment insulinemic index (II), the treatment II by age of horse, overall mean serum insulin area under the curve (mIU/mL/min) divided by the grams of treatment consumed (insulin AUC/g), and the treatment insulin AUC/g by age of horse II (SEM) Treatment Dextrose Whole oats Rolled oats High fat rice bran Pelleted corn Cracked corn Pelleted steamprocessed corn EnergX [expanded 1] EnergX [expanded 2] EnergX [pelleted] EnergX [unprocessed]
Insulin AUC/g (SEM)
Overall Mean e
2-Year-Olds
Mature*
Overall Mean
2-Year-Olds
Mature*
a
100 (46) 217 (46)b 216 (46)b 107 (46)e 372 (46)a 111 (46)d,e 377 (46)a
100 (161) 178 (168) 294 (168) 201 (168) 294 (161) 69 (161) 373 (168)
100 (41) 227 (39)** 197 (39)** 85 (39) 391 (41)** 121 (41) 378 (39)**
1.28 (0.14) 0.53 (0.14)c 0.59 (0.14)c 0.37 (0.14)c 0.89 (0.14)b 0.30 (0.14)c 0.93 (0.14)b
0.63 (0.20) 0.21 (0.21) 0.32 (0.21) 0.23 (0.21) 0.32 (0.20) 0.09 (0.20) 0.42 (0.21)
1.93 (0.21)** 0.86 (0.20)** 0.85 (0.20) 0.51 (0.20) 1.47 (0.21)** 0.51 (0.21) 1.45 (0.20)**
206 (46)b,c,d 184 (46)b,c,d,e 208 (46)b,c 117 (46)c,d,e
277 (168) 243 (161) 161 (168) 73 (161)
189 (39)** 170 (41)** 219 (39)** 128 (41)**
0.51 (0.14)c 0.52 (0.14)c 0.51 (0.14)c 0.49 (0.14)c
0.31 (0.21) 0.28 (0.20) 0.19 (0.21) 0.13 (0.20)
0.71 (0.31) 0.28 (0.20) 0.83 (0.20)** 0.86 (0.21)**
Overall mean treatment insulinemic index (II) were calculated by taking the area under the curve (AUC) of the serum insulin for each treatment divided by the AUC of the dextrose control and multiplying by 100. Parameters are relative to baseline measurements. a,b,c,d,e Means within a column not sharing a common superscript differ (P % .05). * Overall mature different than 2-year-old (P < .01). ** Mature different than 2-year-old within a given treatment (P % .05).
Table 5. Overall mean treatment peak serum insulin (INS) concentrations (mIU/mL), the treatment peak serum INS concentrations by age of horse, overall mean treatment time (min) to peak serum insulin concentrations, and the treatment time to peak serum insulin concentrations by age of horse with the amount consumed used as a covariate for determination of peak INS Peak INS (SEM) Treatment Dextrose Whole oats Rolled oats High fat rice bran Pelleted corn Cracked corn Pelleted steamprocessed corn EnergX [expanded 1] EnergX [expanded 2] EnergX [pelleted] EnergX [unprocessed]
Overall Peak
Time to Peak INS (SEM) 2-Year-Olds
Mature*
b,c
84 (19) 86 (13)b,c 76 (13)b,c 61 (13)c 135 (13)a 51 (13)c 108 (13)a,b
51 (22) 26 (19) 39 (19) 30 (19) 39 (18) 19 (18) 49 (19)
118 (23) 146 (18) 114 (18) 93 (18) 231 (19) 83 (19) 168 (18)
79 (13)b,c 76 (13)b,c 68 (13)c 57 (13)c
31 (19) 33 (18) 22 (19) 31 (19)
128 (18) 119 (19) 115 (18) 82 (19)
Overall Time
2-Year-Olds
Mature*
88 (20) 115 (14)a,b,c 141 (14)a 110 (14)a,b,c 133 (14)a,b 94 (14)c 117 (14)a,b,c
109 (24) 116 (20) 139 (20) 82 (19) 135 (19) 119 (19) 132 (20)
67 (25) 114 (19) 144 (19) 82 (19) 132 (20) 69 (20) 103 (19)
94 (14)c 89 (14)c 140 (14)a 94 (14)b,c
113 (20) 106 (19) 167 (20) 109 (20)
75 (19) 72 (20) 112 (19) 80 (20)
c
Parameters are relative to baseline measurements. a,b,c Means within a column not sharing a common superscript differ (P % .05). * Overall mature different than 2-year-olds (P < .01).
DISCUSSION AND CONCLUSION When designing projects to evaluate glucose and insulin responses in horses, many factors must be considered. Unlike with human beings, no standardized glycemic or II has
been formulated for the equine,15 although Rodiek and Stull16 published a GI of some common equine feeds with the GI of oats being set equal to 100. As would be expected, feeds having higher starch content were found to
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Table 6. Overall mean treatment plasma glucose area under the curve (mg/dL*min) divided by the grams of starch consumed (glucose AUC/g starch), the treatment glucose AUC/g starch by age of horse, overall mean serum insulin area under the curve (mIU/mL*min) divided by the grams of starch consumed (insulin AUC/g starch), and the treatment insulin AUC/g starch by age of horse Glucose AUC/g Starch (SEM)
Insulin AUC/g Starch (SEM)
Treatment
Overall Mean
2-Year-Olds
Mature
Overall Mean
2-Year-Olds
Mature*
Dextrose Whole oats Rolled oats High fat rice bran Pelleted corn Cracked corn Pelleted steamprocessed corn EnergX [expanded 1] EnergX [expanded 2] EnergX [pelleted] EnergX [unprocessed]
NA 1.1 (0.3)b 0.9 (0.3)b 2.4 (0.3)a 0.7 (0.3)b 0.3 (0.3)b 1.1 (0.3)b
NA 1.3 (0.5) 0.9 (0.5) 1.5 (0.5) 0.8 (0.4) 0.3 (0.4) 1.4 (0.5)
NA 0.8 (0.4) 0.9 (0.4) 3.3 (0.4) 0.6 (0.5) 0.3 (0.5) 0.9 (0.4)
NA 1.9 (0.4)a 1.1 (0.4)a,b 1.6 (0.4)a 1.3 (0.4)a,b 0.5 (0.4)b 1.5 (0.4)a,b
NA 0.7 (0.6) 0.6 (0.6) 1.0 (0.6) 0.5 (0.6) 0.1 (0.6) 0.7 (0.6)
NA 3.1 (0.6)** 1.6 (0.6) 2.3 (0.6) 2.1 (0.6)** 0.9 (0.6) 2.2 (0.6)
0.9 (0.3)b 0.9 (0.3)b 0.8 (0.3)b 0.6 (0.3)b
1.0 (0.5) 1.1 (0.4) 0.8 (0.5) 1.2 (0.4)
0.7 (0.4) 0.8 (0.5) 0.7 (0.4) 0.0 (0.5)
1.2 (0.4)a,b 1.4 (0.4)a,b 1.1 (0.4)a,b 1.3 (0.4)a,b
0.7 (0.6) 0.7 (0.6) 0.4 (0.6) 0.3 (0.6)
1.6 (0.6) 2.1 (0.6) 1.8 (0.6) 2.4 (0.6)**
Parameters are relative to baseline measurements. a,b Means within a column not sharing a common superscript differ (P % .05). * Overall mature different than 2-year-old (P < .01). ** Mature different than 2-year-old within a given treatment (P % .05).
have the highest GI. Hoekstra et al used glucose AUC for cracked corn as the response to which other treatments were compared.6 Limitations to using feeds such as oats or corn as the reference are that they can vary both in quantity of starch as well as the availability of that starch. Additionally, rate of consumption of a reference feed can influence results.17 Using dextrose as a control helps to ensure that similar responses can be expected between studies; however, it can be administered both orally and injected intravenously.12 The oral dosage chosen (0.25 g dextrose/kg of BW) was between the 0.2 g dextrose/kg of BW oral dose used by Hoffman et al18 and the 0.3 g dextrose/kg of BW intravenous dose used by the same laboratory.19 This dosage of feed resulted in glycemic indices ranging from 81 to 278 in the current study when horses were fed their dietary treatments at the rate of 1.5 g/kg of BW. Another consideration in choosing the dosage of the dextrose control is that, as an oral drench, the dextrose is immediately consumed, whereas the other treatments are consumed over a period. Hence, even though the amount of dextrose being provided is slightly lower than the amount of starch that was provided in the feed with the lowest starch content (high fat rice bran), the glucose and insulin responses of many of the treatments were similar to the response of the dextrose control, suggesting the dosage may have been appropriate – especially given that only one treatment had peak glucose concentrations and only two treatments had peak insulin concentrations higher than the dextrose control.
The amount of test feedstuff to be administered is another variable that differs between studies. When fed on an equal available carbohydrate basis (2 g carbohydrate/kg of BW), a similar plasma glucose AUC was reported in Thoroughbreds given corn, oat groats, and rolled barley.20 The glucose and insulin peaks, as well as AUC responses, did not vary by processing method in Standardbreds fed 630 g starch per day from oats or corn at a moderate intake (between 1.2 and 1.5 g starch/kg of BW).21,22 By virtue of providing similar amounts of starch, it would reason that detecting differences between treatments would be difficult even with different availability of that starch. Another technique included feeding an amount of feed calculated to provide 4 Mcal digestible energy (DE) per meal.16 Although this technique resulted in differences in glycemic responses, the DE of a feedstuff was calculated from book values and simply represents averages that could vary greatly from the actual DE of the feedstuffs tested. Thus, in this study, it was decided the testing should be done on a standard weight of feed per kg of BW basis. Furthermore, most horse owners provide feed on the basis of the weight of the feedstuffs given, as opposed to starch content or energy content. Although those factors influence how much feed is given, it is the comparison of equal quantities of feeds that tend to be of the most practical benefit for horse owners. Of note, the amount of treatment being administered was based off the weight of each horse from the previous week, although the amount consumed was later calculated
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from the weight determined just before testing with young horses losing an average of 1.3 kg/wk and the mature horses losing an average of 2.2 kg/wk. This resulted in minor variations from the target intake of 1.5 g/kg of BW even when all the feedstuff was consumed. Although most horses consumed all of the treatments offered to them, the high fat rice bran and EnergX [unprocessed] had lower consumption rates. The EnergX [unprocessed] also had the lowest GI. Excluding that treatment, one could argue that the dextrose dosage could be increased given that whole oats, used as the reference in some other studies, had a GI of 136 and only one feed that was entirely consumed had a GI lower than the control. However, it should be pointed out that all of the feeds being tested were concentrates that would be expected to have a high GI – especially compared with forages or some by-products.16 Additionally, a dosage amount of between 0.20 and 0.25 g dextrose/kg of BW represents an amount of glucose somewhat equivalent to the amount often consumed in a grain meal.17 Although the horses did not receive any concentrate other than the test feedstuffs during the course of the study, horses had prior familiarization with eating rolled oats and cracked corn suggesting that a lack of familiarity with unprocessed EnergX and high fat rice bran may have been the reason for incomplete consumption. Glycemic response has been used as an indirect measure of prececal starch digestibility.6 Although attention has been paid to the glycemic response of various feeds to minimize insulin resistance,23 a low response could indicate starch is escaping to the hindgut of the horse - an undesirable consequence. With this in mind, two trials (data not shown) were conducted using ileal cannulated pigs at the Cargill Animal Nutrition Innovation Campus to determine prececal starch digestion of some of the same ingredients, processed identically, used in this trial. The pigs, averaging 41 kg, were fed on an equal starch basis at the rate of 1.9% starch/kg of BW. Pigs were used as a model for equine because of the similar small intestine anatomy and physiology, although a-amylase activity has been shown to be lower in horses than in pigs.7 When data from those trials were combined, the ileal starch digestibility of EnergX pellet (97.0%), EnergX unprocessed (96.2%), EnergX expanded 1 (95.8%), EnergX expanded 2 (94.9%), corn pellet (95.2%), and pelleted steam corn (96.9%) were similar. Other ingredients such as cracked corn (89.8%), whole oats (91.3%), and steam rolled oats (90.4%) had a slightly lower ileal starch digestibility. The data from the horse trial and the pig trial could explain the low GI of the cracked corn because it would rank as one of the lower starch digestibility ingredients used in this pig trial. However, when the corn was processed, the risk of starch leaking to the cecum would decrease because of an increased amount of starch digestibility by about 5%–6%. It also makes sense
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that lower starch ingredients that have equal starch digestibility of higher starch ingredients would have less starch available to leak to the cecum of equine. An example of this can be seen when comparing EnergX pellet versus pelleted steam-processed corn. Both of these ingredients have a starch digestibility of approximately 97% but total starch in the EnergX pellet was 45.6% and in pelleted steamprocessed corn was 64.1%, which leads to an approximate 27% reduction in the amount of starch available to leak into the hindgut. Although age did not have a detectable effect on GI or the glucose AUC/g of feed consumed, there were differences by treatment. Several treatments had a greater GI than did the control. Processing through rolling, pelleting, and expanding increased the GI of the tested feedstuffs, although the ‘‘expanded 2’’ method of processing designed to increase the gelatinization of starch produced only a trend for an increase over the control. This was probably because of the fact that the ‘‘expanded 2’’ product actually had a lower gelatinization of starch relative to the ‘‘expanded 1’’ product. Gelatinization is the irreversible swelling and destruction of the crystalline structure of the starch granules,24 which can result in the starch becoming more available for enzymatic digestion. Although physical processing such as grinding of grains affects digestibility of starch prececally, the effect is not as dramatic as thermal and hydrothermal processing techniques.7 Hoekstra et al reported the highest GI for steam-flaked corn, relative to cracked or ground, and suggested it reflects an increased prececal starch digestibility because of thermal processing.6 This conclusion regarding prececal improvement in starch digestibility of grains because of thermal processing is supported in the pig ileal starch digestibility data presented earlier. In contrast to the processed grains, all unprocessed grains, cracked corn, and the high fat rice bran did not differ from the control in GI, suggesting that the total starch digested in these treatments was lower (as was the case with high fat rice bran which contained only 22.5% starch) or less available to digestion in the foregut. For example, it has been reported that whole corn had lower prececal starch digestibility than oats and also resulted in lower blood glucose concentrations when fed to ponies.25 High fat rice bran had the second highest AUC when standardized by accounting for differences in intake, thus suggesting that the lower GI was due to a reduced consumption rate. By comparison, although horses consumed less EnergX [unprocessed] than the other treatments (excluding the control), when standardized by evaluating the AUC on the basis of amount eaten, it did not differ from the processed EnergX treatments. Although the rate of intake was shown to not correlate with the AUC for blood glucose concentration in a study by Harris et al,26 differences in the amount consumed certainly
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would affect results and need to be considered. The pelleted steam-processed corn continued to elicit the greatest response (excluding the control), whereas the cracked corn produced the lowest when based on the amount consumed. Although peak plasma glucose concentrations were greater with the 2-year-old horses, the difference was not dramatic (roughly 5% difference). The greater peak plasma glucose concentration reported in the pelleted corn and pelleted steam-processed corn treatments presumably reflects a greater availability of starch for prececal digestion. Interestingly, Vervuert et al reported no association of the highest degree of gelatinization in different barley types with the most pronounced glycemic or insulinemic responses.27 The time until peak glucose concentrations was reached was longer with the 2-year-olds than mature horses, which may simply be a reflection of the consumption rate. Pelleted corn and pelleted steam-processed corn exhibited a longer time until peak glucose concentrations, potentially because of the large amount of starch available for digestion. Besides consumption rate, as suggested in this study, Pagan et al demonstrated increasing intake from 0.75 kg up to 1.5 kg increased time to peak glucose concentration.28 Relatively few studies in horses have examined the relationship between glycemic and insulinemic responses15 despite the attention paid to the glycemic response of horses to various feeds to try and control insulin resistance.23 A striking revelation of this study is the difference in insulin response between mature and 2-year-old horses. The mature horses exhibited a much higher insulin response despite having relatively similar glucose concentrations, suggesting that the mature horses had reduced insulin sensitivity. Both the pelleted corn and the pelleted steam-processed corn yielded the greatest insulin response to a meal, as represented by the II, which reflects the high glucose response of both treatments. Whole oats and rolled oats produced the next highest II, which did not differ from the II for the three processed EnergX treatments. As with the plasma glucose concentrations, the high fat rice bran, cracked corn, and EnergX [unprocessed] produced the lowest response, which did not differ from the control. Additionally, the EnergX [expanded 2] treatment did not differ from the control, suggesting the increased starch gelatinization desired in the processing steps did not occur. When the serum insulin AUC was divided by the amount of treatment consumed, the dextrose treatment yielded the highest results. Pelleted corn and pelleted steamprocessed corn were intermediary and the other treatments were the lowest, suggesting a decrease in insulin release with these other treatments. Of note, when the serum insulin AUC was divided by the amount of starch consumed, cracked corn yielded the lowest result, suggesting, once again, a lower digestibility of the starch.
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The peak insulin concentrations were about 4 times greater in the mature horses (127 mIU/mL) than in 2-year-olds (33.6 mIU/mL), despite 2-year-old horses having higher peak plasma glucose concentrations. The treatment differences seen with peak serum insulin concentrations reflect the variations seen in peak plasma glucose concentrations, as do the differences in time to peak serum insulin concentrations with the peak occurring later in the 2-year-olds. This, too, likely reflects the slower feed consumption rate by the 2-year-olds. The elevated insulin response in the mature Arabian horses is an intriguing finding with implications for horse health. It has been suggested that horses on a low-starch diet were less efficient in metabolizing blood glucose than those consuming a grain-based diet.29 Higher glycemic responses would be expected in horses unaccustomed to receiving high GI feeds (such as those on this study) as compared to horses that are adapted to them.18 Additionally, horses consuming only forages would be expected to have decreased rates of glucose clearance compared with ones that are fed high starch concentrates when subjected to a standardized dextrose challenge. Horses may adapt to a diet rich in starch and sugar and the result may be a moderated glucose response. However, insulin sensitivity has also been shown to decrease when horses were fed a diet rich in starch and sugar.19 This can be of great concern when managing obese horses as they can have an increased likelihood of insulin resistance.19 Using the Minimal Model approach to access glucose and insulin dynamics, Treiber et al reported lower insulin sensitivity in ponies with a history of recurrent laminitis on spring pasture.9 In this case, the insulin sensitivity was not related to obesity although ponies have been shown to have a higher degree of insulin resistance than Dutch Warmblood horses.30 In the present study, the mean body condition score did not differ between age groups (2-year-olds ¼ 5.6; mature ¼ 6.4) and did not clearly correlate to the responses of the horses. Although it is important to minimize starch to the equine large intestine by having high prececal starch digestibility, high glucose and insulin responses have been associated with health issues in human beings.31 The same concern likely exists with horses and a controlled response—one in which the glucose and insulin concentrations in the blood are not exaggerated but little starch escapes to the hindgut – potentially may be the ideal goal for which to strive. In populations where insulin insensitivity is an issue, such as in this population of mature Arabians, proper selection of dietary constituents, including the method in which feedstuffs are processed, may aid in preventing various health problems. These data indicate that an insightful awareness of the mode of glycemic response is needed to deliver a diet that will successfully manage the response. A low glycemic
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response is not always best as can be seen in the cracked corn data and the potential for cecal starch leakage to occur. A low glycemic response can deliver a desired response if the starch is nearly completely digested prececally. Also, thermal processing of ingredients can have a major effect on glycemic and insulinemic responses in horses. Although starch and sugar concentrations are an important part of controlling glycemic and insulinemic responses, other nutrients such as gelatinized starch, fat, and fiber play in important role.
14. Wolver TMS, Jenkins DJA, Jenkins AL, Josse RG. The glycemic index: methodology and clinical implications. Am J Clin Nutr 1991; 54:846–854. 15. Vervuert I, Coenen M. Glycaemic index of feeds for horses. Pferdeheilkunde 2005;21:79–82. 16. Rodiek AV, Stull CL. Glycemic index of ten common horse feeds. J Equine Vet Sci 2007;27:205–211. 17. Ralston SL. Factors affecting glucose and insulin metabolism in young horses. Pferdehilkunde 2005;21:83–86. 18. Hoffman RM, Kronfeld DS, Cooper WL, Harris PA. Glucose clearance in grazing mares is affected by diet, pregnancy, and lactation.
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