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Fat Supplementation to Alfalfa Diets for Refeeding the Starved Horse
TheFat Professional Animal (2003):47–54 Supplementation for Scientist Refeeding19 Starved Horses
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to Alfalfa FatDietsSupplementation for Refeeding the Starved Horse C. L. STULL*,1, PAS, P. J. HULLINGER†, and A. V. RODIEK‡, PAS *University of California, Davis, CA 95616; †California Department of Food and Agriculture, Sacramento, CA 95814; and ‡California State University, Fresno, CA 93740
Abstract
thus indicating hypophosphatemia. Serum magnesium increased in horses The objective was to examine the consuming the AH diet, whereas concenmetabolic responses of chronically starved trations in horses consuming the AO diet horses using two isoenergetic diets consistently were below reference range, consisting of either 100% alfalfa hay leading to mild hypomagnesemia. (AH) or 80% alfalfa hay with 20% corn Hypokalemia did not develop in either oil (AO), on a caloric basis. Two 10-d treatment group, as all serum potassium trials were conducted using 14 emaciated, concentrations remained in the normal but otherwise healthy, horses with an reference range. The substitution of corn initial mean body condition score of 1.8 ± oil in isoenergetic alfalfa diets was not 0.9 and mean BW of 336 ± 31 kg. The advantageous in minimizing or preventinitial DE intake of 50% of maintenance ing hypophosphatemia or hypowas gradually increased to 100% during magnesemia for refeeding starved horses. the 10-d refeeding period. Two of the The AH diet is recommended as more horses receiving the AH diet were supportive in refeeding starved horses euthanized, thus 12 horses (AH, n = 7; because of the greater intake of dietary AO, n = 5) survived the 10-d study. One phosphorous and magnesium. preprandial and four postprandial venous blood samples were collected daily. (Key Words: Equine, MalnourishDietary effects (P<0.01) were shown for ment, Starvation, Insulin, Phosphoblood serum or plasma free fatty acids rus.) (FFA), phosphate, magnesium, and 2,3diphosphoglycerate (2,3-DPG), but not glucose, insulin, or potassium concentraChronically starved horses are tions. The response of insulin and FFA during the 10-d study showed differences similar to emaciated humans in their response to refeeding; they experience (P<0.05) between AH and AO diets. a broad range of metabolic derangeSerum phosphate concentration in horses ments including severe hypoon both diets exhibited a general slow decline with concentrations below normal magnesemia and hypophosphatemia with a decrease in 2,3reference range during d 5 through 10, diphosphoglycerate (2,3-DPG) concentration, which is important in the 1To whom correspondence should be adhemoglobin-oxygen association in dressed: [email protected] the red blood cell (Witham and Stull,
Introduction
1998). Anecdotal reports suggest that abrupt refeeding of horses may cause death within 3 d (Kronfeld, 1993). Limited reports have evaluated practical diets for refeeding chronically starved horses (Kronfeld, 1993; Finocchio, 1994). Our earlier study on refeeding starved horses showed that isoenergetic diets composed of either alfalfa or oat hay produced lower insulin and glucose responses than an isocaloric diet consisting of a combination of oat hay and a commercial complete feed (Witham and Stull, 1998), a result consistent with clinical experiences (Kronfeld, 1993; Finocchio, 1994). The benefits of corn oil supplementation in equine diets have been well documented over the last decade. One benefit to the starved horse is that the postprandial response of insulin in healthy horses is less with the addition of corn oil, which may be advantageous to lessen hypophosphatemia and hypomagnesemia frequently found with the refeeding syndrome (Stull and Rodiek, 1988). Thus, the objective of this study was to examine the metabolic responses of chronically starved horses during refeeding using two isoenergetic diets consisting of either 100% alfalfa hay (AH) or 80% alfalfa hay with 20% corn oil (AO), on a caloric basis.
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Materials and Methods
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treatment, and five horses were assigned the AO diet. Horses had free Horses and Study Design. All access to water throughout the study, phases of the study were approved by but no supplemental salt or minerals the University of California Animal were available. Care and Use Committee. Two 10-d Alfalfa hay samples from each trial feeding trials were conducted in 1996 were analyzed by an AOAC-approved and 1999 using 14 mature horses. laboratory (JL Analytical Service, Emaciated horses of mixed light Modesto CA) prior to the start of breeds (8 mares, 5 geldings, and 1 each trial (Table 1). The AO diet was stallion) and unknown histories were formulated with 80% of the calories purchased in Mexicali, Mexico or from alfalfa hay and 20% of the Albuquerque, New Mexico by a calories from corn oil; hence, intake commercial buyer. Horses were was calculated from the known transported for approximately 8 h by composition of these two ingredients trailer to Davis, California. Horses (Table 2). The following equation for ranged in age from 2 to 15 yr (8.3 ± maintenance was used to calculate 4.9 yr), as estimated by examination the daily DE requirement (Mcal/d) for of teeth, and weighed between 265 each horse; “target” BW was estiand 373 kg (336 ± 31 kg) on arrival. mated to be 125% of each horse’s BW Mean height at the withers was 144 ± at the start of the trial: Mcal DE/d = 4.8 cm. Body condition scores (BCS) 1.4 + 0.03 BW (NRC, 1989). ranged from 1 to 4 (1.8 ± 0.9), using All feed was withheld on arrival (d a maximum score of 9 (Henneke et 0), and the first meal was fed on the al., 1983). Horses in each refeeding following morning (d 1) at 0900 h. trial (Trial 1, n = 7; Trial 2, n = 7) were The DE per day gradually increased emaciated but clinically free of overt from 50% divided into six meals disease, as determined by physical offered at 4-h intervals (0900, 1300, examination. 1700, 2100, 0100, and 0500 h) on d 1 The same barn was used for each to 3, to 75% of daily DE divided into trial to house the horses for the 10-d six meals on d 4 to 5. On d 6 to 10, trial. Approximately 40% of the floor 100% of the DE per day was fed in in each concrete stall (6.8 × 3.5 m) three feedings at 8-h intervals (0900, was covered by rubber mats, and no 1700, and 0100 h). Meals were bedding was provided to eliminate the possibility that horses would TABLE 1. Composition of alfalfa consume the bedding. Horses were hay (AH) fed during Trial 1 weighed daily between 1300 and 1400 (1996) and 2 (1999) and alfalfa h using an electric portable scale hay supplemented with corn oil (Model H90-3042; Fairbanks, St. (AO) fed during Trial 2 (1999). Johnsbury, VT). Diet Formulation and Feeding Trial 1 Trial 2 Protocol. Horses were assigned to one of the two following isoenergetic Item AH AH AO diets: 100% alfalfa hay (AH; n = 9) or 80% alfalfa hay and 20% corn oil DE, Mcal/kg 2.28 2.48 3.78 (AO; n = 5 ). Because the availability Moisture, % 11.9 10.5 8.4 of a sufficient number of emaciated CP, % 21.4 20.0 16.0 horses was limited to meet research Crude fiber, % 22.1 24.9 20.0 objectives and schedules, data from Crude fat, % 2.3 3.4 23.4 horses in a previous study (1996) were Ash, % 7.5 7.2 5.8 combined with Trial 2. Feeding Phosphorus, % 0.44 0.24 0.19 protocols, sampling schedules, and Magnesium, % 0.62 0.46 0.37 housing were replicated between Potassium, % 1.8 1.7 1.4 Calcium, % 0.92 0.70 0.56 trials. All horses in Trial 1 were Sodium, % 0.21 0.11 0.09 assigned the AH treatment. In Trial 2, two horses were assigned the AH
individually weighed for each horse, and all orts were collected, weighed, and recorded prior to the next meal. Blood Collection. On the day of arrival, a 14-ga indwelling catheter was placed aseptically in the left jugular vein of each horse. If functional problems or abnormal swelling occurred at the catheter site, a replacement catheter was inserted in the opposite jugular vein several hours prior to the next blood sampling period to avoid any stress responses caused by catheterization procedures. Each day of the 10-d trial, preprandial blood samples (30 mL) were collected at 0845 h, prior to the first meal (0900 h). Horses were fed their assigned meal at 0900 h. Postprandial samples were drawn every hour starting at 1000 h and continuing until 1300 h for a total of four samples. Blood samples to be analyzed for electrolytes, FFA, insulin, and glucose concentrations were immediately placed on ice and allowed to clot. Serum was obtained from these samples and frozen at –56°C. Additionally, blood samples were collected in evacuated tubes containing potassium oxalate and sodium fluoride for the determination of 2,3DPG concentration. One milliliter of this blood was added to 3 mL of cold trichloroacetic acid and centrifuged; the supernatant was then separated and frozen for storage. Selected electrolyte (magnesium, inorganic phosphate, and potassium) concentrations were quantified using an automated spectrophotometry system (Ectachem DT-60; Eastman Kodak Co., Rochester, NY). Free fatty acids and 2,3-DPG concentration were measured by enzymatic colorimetric assays (Procedure 541®; Sigma Diagnostics, St Louis MO and Procedure 337®; Boehringer-Mannheim, Indianapolis, IN, respectively). Glucose concentration was determined by use of an autoanalyzer (YSI 2300 STAT Plus®;Yellow Springs, OH). Insulin concentration was analyzed using a commercially available radioimmunoassay kit (Kit D1804®; Micromedic Systems, Horsham, PA)
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Bacteriologic cultures were positive for Salmonella saint-paul. A second mare (17 yr; BCS, 1.5) developed mild diarrhea on d 5 while exhibiting behavior consistent with increasTrial 1 Trial 2 ing abdominal pain, which became uncontrollable; euthanasia was Item AH AH AO elected on d 8. Necropsy revealed volvulus of the left large colon at the diaphragmatic flexure. It is Offered DE, Mcal/d 6.59 7.31 7.09 unknown if the AH diet contributed Offered feed, kg/d per horse 2.89 2.95 1.87 Consumed feed, kg/d per horse 2.84 2.90 1.78 specifically to the cause of death, as CP, g/d per horse 609 580 285 the number of horses that were Crude fiber, g/d per horse 628 722 355 euthanized was small, and the Crude fat, g/d per horse 65 99 417 causes of death were seemingly Ash, g/d per horse 213 209 103 unrelated. Only data from the 12 Phosphorus, g/d per horse 12.5 7.0 3.4 horses (AH diet, n = 7; AO diet, n = Magnesium, g/d per horse 17.6 13.3 6.6 5) that survived the 10-d study are Potassium, g/d per horse 51.1 49.3 24.2 included in the statistics. Calcium, g/d per horse 26.6 20.3 10.0 Two different sources of alfalfa Sodium, g/d per horse 6.0 3.2 1.6 hay were used for Trials 1 and 2. The nutrient content of each of the two hays (Table 1) was typical for validated for equine blood by Reimers assumptions of normality and high quality alfalfa hay (NRC, 1989). et al. (1982). homoscedasticity were examined A preliminary statistical analysis Statistical Analyses. Repeated prior to finalizing the analysis, using compared the responses of horses on measures ANOVA (PROC GLM; SAS Wilk-Shapario (Wilk and Shapiro, the AH treatment from 1996 and Inst. Inc., Cary, NC) was used to 1968) and Levene tests (Levene, 1960). 1999 to determine whether it was examine the effects of trial, time Variables that required transformaappropriate to combine these groups (daily), and diet on daily blood tion (using logs) were analyzed as in a single analysis. Those results are responses of each horse to the meal logs. Significance is claimed whenshown in Table 3. There were tempooffered at 0900 h. For the parameters ever P<0.05. ral (day-to-day) responses (P<0.05) for of glucose, insulin and FFA, the all variables except 2,3-DPG and magnitude of the postprandial potassium, and trial effects (P<0.05) increment response from the were shown for FFA, 2,3-DPG, magneThe horses used in this study were preprandial (0845 h) measurement sium, and inorganic phosphate. purchased in an emaciated stage of was calculated as the area under the However, because there were no starvation, but were otherwise curve using the trapezoidal method significant time × trial interactions healthy, with no signs of disease or and log-transformed data. For 2,3(P>0.05 for all parameters), indicating lack of dental function. The poor DPG concentration and serum similar responses of the parameters condition of these horses was probelectrolytes, including inorganic during the 10-d study, the data from ably due to economic hardship phosphate, magnesium, and potasthe trials were combined for the AH experienced by the owner or unavail- diet (n = 7). sium, repeated measures ANOVA was based on log-transformed data using able forage during drought condiThe primary aim of this study was a weighted mean. For these variables, tions. The horses were representative to determine the metabolic responses postprandial peaks were not expected; of cases presented to humane officers, of starved horses to two isoenergetic nutritionists, veterinarians, and therefore, these were measured only diets with and without the inclusion rescue shelter personnel for rehabilionce or twice following the meal. of corn oil. As expected, the protein, tation. All horses were ambulatory The Huynh-Feldt adjustment was fiber, and mineral content was less in used with the univariate results as an following transport from the site of the AO diet as compared with the AH purchase. Two of the 14 horses in adjustment for serial correlation of diet (Table 2) because of the nutrithe residual (Huynh and Feldt, 1976). Trial 1 were euthanized prior to the tional dilution caused by the addiend of the 10-d study. A 7-yr-old Post-hoc analyses were conducted tion of corn oil. Because of the desire mare (BCS, 2.5) assigned the AH diet using pairwise contrast tests (for to examine practical equine diets, no refused to eat after the first meal, mean effect of diet) or contrast effort was made to formulate these developed profuse watery diarrhea on diets on an iso-nitrogenous or other decomposition (for time × diet d 2, and was euthanized on d 4. interactions). For all analyses, the nutrient-equivalent basis.
TABLE 2. Calculated daily feed and nutrient intakes of alfalfa hay (AH) during Trial 1 (1996) or Trial 2 (1999) or of alfalfa hay and corn oil (AO) during d 1 to 3 (50% DE/d).
Results and Discussion
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pared with other common feeds including both forages and concentrates. There was an approximate twofold difference in CP intake between the diets (Table 2); thus, the amino acid intake also was doubled Item P (Trial) P (Time) P (Time x trial) with the consumption of the AH diet. This increased intake of amino Glucose 0.1698 0.0189 0.1242 acids likely contributed to the greater Insulin 0.1350 0.0009 0.1596 response of insulin from the AH diet Free fatty acids 0.0281 <0.0001 0.2987 during the 10-d study. Interestingly, Phosphate 0.0340 <0.0001 0.3201 arginine-induced insulin release 2,3-diphosphoglycerate 0.0136 0.7397 0.6349 occurred more consistently in mares Magnesium 0.0091 0.0003 0.4401 than in geldings and stallions Potassium 0.1067 0.3278 0.6701 (Sticker et al., 2001). The number of horses in this refeeding study was too limited to examine responses because Feed Consumption and BW Gain. tion was significant (P<0.0001), of gender, but gender may be a A general decline in BW occurred especially as the DE per meal inpotential factor in manipulating between d 1 and 5 when 50 to 75% of creased during d 6 to 10 (Table 4; insulin response through dietary the daily DE requirement was offered; Figure 1). No difference (P=0.17) in mechanisms. small increases in BW were shown insulin response was shown between Concentrations of glucose exhibbetween d 6 and 10 when 100% of diets; however, the interaction of day ited a daily postprandial fluctuation the daily DE requirement was fed and diet (time × diet) was significant within each diet (Figure 1), but there (Figure 1). No difference (P>0.05) in (P=0.03), with the mean insulin was no difference (P=0.98) in the BW response was observed between response of the AH diet exhibiting glucose response between diets over diets during the 10-d study period consistently greater concentrations the 10-d study (time × diet) (Table 4). (Figure 1). The mean BW on arrival than the AO diet over the 10-d study The very low soluble carbohydrate and d 10 was 325 ± 35.6 and 325 ± (Table 4). content of both diets along with the 33.9 kg for horses fed the AH diet and In other studies on fed healthy effect of insulin accounts for the lack 340.7 ± 20.8 and 334 ± 22.1 kg for horses, the roughage-to-concentrate of difference (P=0.99) in serum horses fed the AO diet, respectively. ratio of the diet has affected the glucose response between the diets It was not expected that BW gain insulin response, but mainly because during the 10-d study. On a daily would occur during the 10-d study, of the carbohydrate content of the basis, the maximum glucose concenespecially because only 50 to 75% of concentrate portion (Stull and tration was reached 2 to 3 h followthe daily DE requirement was offered Rodiek, 1988; Powell et al., 2000). ing the morning meal. Mean poston d 1 to 5. The palatability of both The substitution of 20% of dietary prandial glucose concentration did diets by the starved horses was calories from alfalfa hay to corn oil not exceed the reference of 99 ± 0.1 evidenced by a consumption of 98.4 in the AO diet reduced the soluble mg/dL reported in healthy horses fed ± 2.7 and 95.1 ± 4.6% of the offered carbohydrate component of the diet alfalfa diets (Stull and Rodiek, 1988). AH and AO diets, respectively, even and might have contributed to the FFA Concentration. Fats are with the inclusion of a high portion lower insulin response in the AO commonly added to equine diets as a of corn oil (20% of the total calories) treatment. However, the soluble source of energy and contribute in the AO diet. carbohydrate component of alfalfa is about three times more DE than the Insulin and Glucose Response. very low in starch (1.6%) (Anon., same weight of grain (NRC, 1989). The mean preprandial insulin con1971), so the reduction effect in the Usually fat is added at about 5 to centrations in horses fed either diet soluble carbohydrates with the 10% of the total diet, but may be were generally comparable to the substitution of corn oil would be supplemented up to 20% of the diet concentration of 4.7 ± 1.0 µU/mL minimal. on a DM basis. Plant sources of fats previously reported (Stull and Rodiek, Infusion of the amino acids lysine such as corn oil, peanut oil, and 1988). The daily response of insulin and arginine, but not aspartic acid or soybean oil are up to 94% digestible concentration following a meal was glutamic acid, have been shown to (Rich et al., 1981). Corn oil was similar to the pattern of the glucose stimulate the release of insulin in selected for this study because of its response, with peak insulin concenhorses (Sticker et al., 2001). Alfalfa wide availability for purchase by trations generally reaching 3 to 4 h has a comparatively high content of horse owners and acceptable palatfollowing the meal. A temporal both lysine (0.90%) (NRC, 1989) and ability by horses (Holland et al., response (day) of insulin concentraarginine (0.79%) (NRC, 2001) com1993). In an earlier equine study
TABLE 3. Repeated measures of log-transformed blood concentration data of the alfalfa diet for Trial 1 (1996) and Trial 2 (1999) (based on daily means). The reported P values reflect a Huynh-Feldt adjustment for autocorrelation among the daily means.
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Figure 1. Mean BW and concentrations of glucose, insulin, FFA, inorganic phosphorus, 2,3-DPG, magnesium and potassium during d 1 to 3 (50% DE/d fed), d 4 to 5 (75% DE/d fed), and d 6 to 10 (100% DE/d fed) in treatments of alfalfa hay (AH, n = 7) and alfalfa hay and corn oil (AO, n = 5).
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using healthy horses, the postprandial insulin response was dampened when oil was added to a single meal of corn (Stull and Rodiek, 1988). A reduction in insulin release is proposed to be advantageous in controlling the intracellular shift of phosphorus, which may contribute to a hypophosphatemic state during the initial refeeding period of starved horses. The serum concentration of FFA showed daily responses, dietary differences, and differences between the diets (P<0.001) during the 10-d study (time × diet) (Table 4). The concentration of FFA showed a rapid temporal decrease following the ingestion of the first meal on d 1 (Figure 1). Distinctly high FFA concentrations in preprandial samples were found in the AO treatment throughout the 10-d study. This result is in contrast to the AH group, which displayed elevated preprandial FFA concentrations only during d 1 to 5, with elevated values diminishing and subsequently disappearing during d 6 to 10 when 100% of the daily DE was offered in three meals per day. The range in preprandial FFA concentration in the horses receiving the AO treatment was 0.15 to 0.28 mmol/L during d 6 to 10, with all mean postprandial FFA concentrations measured at <0.15 mmol/L. In contrast, the mean preand postprandial FFA concentrations in horses receiving the AH treatment during d 6 to 10 ranged from 0.02 to 0.07 mmol/L, which is less than the preprandial FFA concentration of 0.25 ± 0.12 mmol/L reported for healthy resting horses (Stull and Rodiek, 1995). The “peak-and-valley” pattern shown in FFA concentration in the AO-fed horses might have been caused by several factors, including rate of digestion and metabolism of nutrients. The total feed offered from the AO diet was 60% less than that offered from the AH diet, with the oil component of the diet known to be highly digestible (Rich et al., 1981). The rapidly available energy from the AO diet apparently reduces
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the need for FFA mobilization from adipose tissue, thereby resulting in a decline in circulating FFA concentrations. However, the readily digestible and absorbed dietary fat is also rapidly cleared (within several hours) from the blood. This relatively rapid appearance and disappearance of dietary fat from the blood dissipates several hours after the previous meal, with little or no dietary fat sources in the preprandial blood sample collected at 0845 h. During this time, the energy needs of the body tissues are likely met by FFA mobilized from stores in the adipose tissue. Thus, the “valley-and-peak” availability of the dietary energy from the AO diet may explain an inverse “peak-and-valley” variation in serum FFA concentrations. Compared with the AO diet, the AH diet contained no rapidly available energy source. Alfalfa hay is low in both fat and soluble carbohydrates (Anon., 1971; NRC, 1989) and is digested largely by microbial fermentation in the large intestine. Slow microbial fermentation results in a lower but more constant release of VFAs into circulation, which is exhibited by much less fluctuation in the serum FFA concentration profile, especially during d 6 to 10. It has been reported that mares fed diets containing only 50% of required energy and protein for 7 d showed reduced plasma glucose and insulin concentrations, while FFA concentration rose throughout the period. When switched to the control diet providing 100% energy
and protein requirements, glucose and insulin concentrations returned to control levels within 1 h postprandially, and FFA declined to control levels within 2 h of consuming the diet (Sticker et al., 1995). This sharp postprandial decrease in FFA is similar to the response of FFA to the initial meal in both the AH and AO diets of the starved horses. Increases in FFA have also been reported in fasting ponies (Beatz and Pearson, 1972; Naylor et al., 1980; Bauer, 1983), swine (Baetz and Mengeling, 1971) and lambs (Cole et al., 1988). Because glycogen stores are among the first energy stores to be depleted in starved animals, the enhanced oxidation of FFA provided in the AO diet may inhibit glucose uptake and glycolysis by skeletal and heart muscle, leaving glucose for energy needs of the brain and other vital tissues. Selected Serum Electrolytes. During refeeding of chronically starved humans, energy supplied by carbohydrates stimulates the release of insulin. Subsequently, there is influx into the cells of glucose, water, phosphorus, potassium, and other electrolytes. Hypophosphatemia results because of the depletion of total body phosphorus during starvation and an influx of phosphorus into the cells during refeeding. Severe hypophosphatemia in humans produces significant reduction in intracellular phosphate metabolites in red blood cells, such as ATP and 2,3-DPG, which limits cell metabolism
TABLE 4. Repeated measures analysis of log-transformed data for the comparison of the AH and AO diets (based on daily means). The reported P values reflect a Huynh-Feldt adjustment for autocorrelation among the daily means. Item
P (Diet)
Glucose Insulin Free fatty acids Phosphate 2,3-diphosphoglycerate Magnesium Potassium
0.9125 0.1745 0.0010 0.0486 0.0003 0.0118 0.0986
P (Time) 0.0006 <0.0001 <0.0001 <0.0001 0.0041 0.0029 0.0847
P (Time x diet) 0.9876 0.0346 0.0006 0.4028 0.2449 0.0825 0.9519
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Fat Supplementation for Refeeding Starved Horses
and survivability. This, in turn, leads to increased hemoglobin and oxygen affinity, followed by the impaired release of oxygen delivery to tissues (Lichtman et al., 1971; Knochel, 1977). Cardiac, respiratory, hematological, and neuromuscular compromise follows (Solomon and Kirby, 1990). Isocaloric diets fed to starved horses showed that serum phosphorous concentration decreased dramatically during the first 5 d of refeeding, predisposing the horses to hypophosphatemia (Witham and Stull, 1998). The response of serum inorganic phosphate, 2,3-DPG, and potassium concentrations during the 10-d study were similar (time × diet; P>0.05; Table 4) between the AH and AO diets. The AO group exhibited consistently greater (P<0.05) serum inorganic phosphate concentrations than the horses fed the AH diet (Figure 1). Phosphate concentration showed daily responses (P<0.0001) to the ingestion of a meal (Table 4). The phosphorous concentration of both diets showed similar responses (time × diet, P=0.40) during the 10-d study, with a general slow decline in mean concentration during the study. Mean phosphorous concentration declined below a normal reference range of 3.1 to 5.6 mg/dL (Carlson and Smith, 1990) on d 3 and 5 in horses fed the AH and AO diets, respectively, indicating the onset of a hypophosphatemic condition. The dietary phosphorous intake of the AH group was two- to threefold greater than the AO group, but no difference was shown (P>0.05) between diets in the response of serum phosphate caused by increased intake. Similarly, whole blood 2,3DPG concentrations in each diet were below normal reference range for healthy horses at the initiation of refeeding and exhibited slight decreases throughout the 10-d study, but no differences (P=0.24) existed between diets over time (Table 4). The concentration of 2,3-DPG showed daily responses (P=0.004) to the ingestion of the meal, and greater mean concentrations of 2,3-DPG were
shown (P=0.0003) consistently in the AH treatment group (Table 4; Figure 1). Mean concentrations at all sampling points in both dietary treatments were below reference resting concentration for healthy horses (2.2 ± 0.2 µmol/mL) (Stull, 1983); values ranged from 0.5 to 1.1 and 1.8 to 1.3 µmol/mL for the AO and AH groups, respectively. A decrease in the production of red blood cell 2,3-DPG has been reported in human refeeding following severe weight loss as a consequence of severe hypophosphatemia (Lichtman et al., 1971; Solomon and Kirby, 1990), and the declining 2,3-DPG concentrations during refeeding in horses appear to support this physiological complication of severe hypophosphatemia. Both diets throughout the 10-d study exceeded the nutrient requirement of potassium for maintenance, which is 20 g/d for a 400-kg horse (NRC, 1989). Serum potassium concentration showed a decrease at the initiation of refeeding in each of the diets, with concentration increasing during the remainder of the 10-d study. The twofold difference in dietary potassium intake between the diets did not affect the response of serum potassium concentration, which was similar (P=0.95) between the diets during the 10-d study (Table 2). Even though potassium concentration was consistently greater in the AO horses, no dietary (P=0.09) or daily effects (P=0.08) were observed (Figure 1). Hypokalemia was not present in horses of either treatment group, as all mean serum potassium concentrations were within the normal reference range (2.4 to 4.7 mmol/L) (Brobst and Parry, 1987). Magnesium Concentration. Hypomagnesemia has been documented as producing synergistic deleterious clinical effects along with hypophosphatemia during the refeeding period in human patients (Holcombe et al., 1985; Vanlandingham et al., 1981). Normal reference range for magnesium concentration in healthy mature horses is 1.3 to 2.5 mg/dL (Brobst and Parry, 1987), and all magnesium
concentrations of the AH horses increased slightly throughout the study (1.3 to 2.3 mg/dL) and remained within the reference range during the study. In contrast, the magnesium concentration of the AO group was either at the low end or below reference range, indicating mild hypomagnesemia. This was reflective of the 2- to 2.5-fold greater daily intake of dietary magnesium (Table 2) in the AH group. An earlier study with starved horses offered a diet of alfalfa hay showed increasing serum magnesium concentrations that were not evident in the subjects fed either oat hay or a combination diet, which had a lower magnesium concentration than the alfalfa diets (Witham and Stull, 1998). Dietary effects (P=0.012) confirmed magnesium concentration was generally less in the AO group compared with the AH group throughout the study (Table 4; Figure 1). The daily effect (P=0.003) of magnesium concentration was shown as the postprandial sample was consistently greater in magnesium concentrations as compared with the preprandial sample, especially during d 6 to 10. The response between diets during the 10d study (time × diet) tended (P=0.08) to be greater in the AH group as compared with the AO treatment. The substitution of corn oil for alfalfa does not appear to be advantageous in minimizing or preventing hypomagnesemia during refeeding.
Implications Hypophosphatemia developed during the 10-d refeeding period for horses in both the AH and AO groups, even though the insulin effect was dampened by the substitution of corn oil in the AO diet. Additionally, mild hypomagnesemia was associated with the AO diet; whereas, the serum magnesium levels in the AH diet increased during the 10-d study. The interrelationship of hypomagnesemia with severe hypophosphatemic condition of the AO diet may produce synergistic deleterious effects; thus, the AH diet
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is recommended as more supportive of the refeeding of the starved horse because it provides a greater intake of dietary phosphorous and magnesium. Nutrient analyses of feeds should be performed in practical settings for the nutritional rehabilitation of starved horses to ascertain the levels of important nutrients, especially phosphorus and magnesium. Future research should examine the effects of supplemental sources of magnesium and phosphorus during the refeeding of starved horses.
Acknowledgments The project was supported by the Center for Equine Health with funds provided by the Oak Tree Racing Association, the State of California Pari-Mutuel Fund, and contributions by private donors. The authors also thank Purina Mills Inc. for additional funding of the research, the pathologists at California Animal Health and Food Safety Laboratory System for performing the necropsy procedures, Neil Willits for statistical assistance, and Kelly Weaver and Marsha Feldman for laboratory analyses. The assistance of Sheila Bruce, Angela Hermann, Kelsie Luiz, Lisa Machado, and Chad Stewart is greatly acknowledged for the care of the horses.
Literature Cited Anon. 1971. In Atlas of Nutritional Data on United States and Canadian Feeds. p 9 and 251. National Research Council, National Academy of Sciences, Washington, DC. Baetz, A. L., and W. L. Mengeling. 1971. Blood constituent changes in fasted swine. Am. J. Vet. Res. 32:1491. Baetz, A. L., and J. E. Pearson. 1972. Blood constituent changes in fasted ponies. Am. J. Vet. Res. 33(10):1941.
Bauer, J. E. 1983. Plasma lipids and lipoproteins of fasted ponies. Am. J. Vet. Res. 44:379. Brobst, D. F., and B. W. Parry. 1987. Normal clinical pathology data, Table 4. In Current Therapy in Equine - 2. N. E. Robinson (Ed.). p 728. W. B. Saunders Co, Philadelphia, PA. Carlson, G. P., and B. P. Smith. 1990. Clinical chemistry tests. In Large Animal Internal Medicine. (1st Ed). B. P. Smith (Ed.) p 390. C. V. Mosby Co., St. Louis, MO. Cole, N. A., C. W. Purdy, and D. M. Hallford. 1988. Influence of fasting and postfast diet energy level of feed intake, feeding pattern and blood variable of lambs. J. Anim. Sci. 66:798. Finocchio, E. J. 1994. Equine starvation: Recognition and rehabilitation of the recumbent, malnourished horse. Large Anim. Vet. 49:6. Henneke, D. R., G. D. Potter, J. L. Kreider, and B. F. Yeates. 1983. Relationship between body condition score, physical measurements and body fat percentage in mares. Equine Vet. J. 15:371. Holcombe, B. J., and M. M. Adams. 1985. Metabolic complications associated with enteral nutritional support. Nutr. Support Serv. 5:26. Holland, J. L., T. N. Meacham, D. S. Kronfeld, and W. Tiegs. 1993. Acceptance of lecithin containing diets by horses. In Proc.13th Equine Nutr. Physiol. Symp., Gainesville, FL. p 54. Equine Nutr. Physiol. Soc., Savoy, IL. Huynh, H., and L. S. Feldt. 1976. Estimation of the box correction for degrees of freedom from sample data in the randomized block and split plot designs. J. Educ. Stat. 1:69. Keys, A., J. Brozek, A. Henschel, O. Mickelsen, and H. L. Taylor. 1950. The Biology of Human Starvation. Vol. 1-2. Univ. Minnesota Press, Minneapolis, MN. Knochel, J. P. 1977. The pathophysiology and clinical characteristics of severe hypophosphatemia. Arch. Intern. Med. 137:203. Kronfeld, D. S. 1993. Starvation and malnutrition of horses: Recognition and treatment. In Proc. First Int. Conf. Equine Rescue. J. Equine Vet. Sci.. 13:298. Levene, H. 1960. Robust tests for equality of variances. In Contributions to Probability and Statistics; Essays in Honor of Harold Hotelling. (1st Ed.). I. Olkin (Ed). p 278. Stanford University Press, Stanford, CA. Lichtman, M. A., D. R. Miller, J. Cohen, and C. Waterhouse. 1971. Reduced red cell glycolysis, 2,3-diphosphoglycerate and adenosine triphosphate concentration, and increased hemoglobin-oxygen affinity caused by hypophosphatemia. Ann. Int. Med. 74:562. National Research Council. 1989. Nutrient Requirements of Horses. (5th Ed.) National Academy Press, Washington, DC.
National Research Council. 2001. Nutrient Requirements of Dairy Cattle. (7th Ed.) National Academy Press, Washington, DC. Naylor, J. M., D. S. Kronfeld, and K. Johnson. 1980. Fasting hyperbilirubinemia and its relationship to free fatty acids and triglycerides in the horse. In Proc. Soc. Exp. Biol. Med. 165:86 Powell, D. M., L. M. Lawrence, B. P. Fitzgerald, K. Danielson, A. Parker, P. Siciliano, and A. Crum. 2000. Effect of short term feed restriction and calorie source on hormonal and metabolic responses in geldings receiving a small meal. J. Anim. Sci. 78:3107. Reimers, J. T., R. G. Cowan, J. P. McCann, and M. W. Ross. 1982. Validation of rapid solidphase radioimmunoassay for canine, bovine and equine insulin. Am. J. Vet. Res. 43:1274. Rich, G. A., J. P. Fontenot, and T. N. Meacham. 1981. Digestibility of animal, vegetable, and blended fats by equine. In Proc. 7th Equine Nutr. Physiol. Symp., Warrenton, VA. p 30. Equine Nutr. Physiol. Soc., Savoy, IL. Solomon, S. M., and D. F. Kirby. 1990. The re-feeding syndrome: A review. J. Parenter. Enteral. Nutr. 14:90. Sticker, L. S., D. L. Thompson, Jr., J. M. Fernandez, L. D. Bunting, and C. L. Pew. 1995. Dietary protein and(or) energy restriction in mares: Plasma growth hormones, IGF-I, prolactin, cortisol, and thyroid hormones responses to feeding, glucose, and epinephrine. J. Anim. Sci. 73:1424. Sticker, L. S., D. L. Thompson, Jr., and, L. R. Gentry. 2001. Pituitary hormone and insulin responses of infusion of amino acids and Nmethyl-D,L-aspartate in horses. J. Anim. Sci. 79:735. Stull, C. L. 1983. Blood parameters of horses trained on a treadmill. Ph.D. Dissertation, University of Illinois, Urbana-Champaign, IL. Stull, C. L., and A.V. Rodiek. 1988. Responses of blood glucose, insulin, and cortisol concentrations to common equine diets. J. Nutr. 118:206. Stull, C., and A. Rodiek. 1995. Effects of post prandial interval and feed type on substrate availability during exercise. Equine Vet. J. Suppl. 18:362. Vanlandingham, M. D., S. Simpson, P. Daniel, and S. R. Newmark. 1981. Metabolic abnormalities in patients supported with enteral tube feeding. J. Parenteral. Enteral. Nutr. 5(4):322. Wilk, M. B., and S. S. Shapiro. 1968. The joint assessment of normality of several independent samples. Technometrics 10:825. Witham, C. L., and C.L. Stull. 1998. Metabolic responses of chronically starved horses to refeeding with three isoenergetic diets. J. AVMA 212(5):691.
47
to Alfalfa FatDietsSupplementation for Refeeding the Starved Horse C. L. STULL*,1, PAS, P. J. HULLINGER†, and A. V. RODIEK‡, PAS *University of California, Davis, CA 95616; †California Department of Food and Agriculture, Sacramento, CA 95814; and ‡California State University, Fresno, CA 93740
Abstract
thus indicating hypophosphatemia. Serum magnesium increased in horses The objective was to examine the consuming the AH diet, whereas concenmetabolic responses of chronically starved trations in horses consuming the AO diet horses using two isoenergetic diets consistently were below reference range, consisting of either 100% alfalfa hay leading to mild hypomagnesemia. (AH) or 80% alfalfa hay with 20% corn Hypokalemia did not develop in either oil (AO), on a caloric basis. Two 10-d treatment group, as all serum potassium trials were conducted using 14 emaciated, concentrations remained in the normal but otherwise healthy, horses with an reference range. The substitution of corn initial mean body condition score of 1.8 ± oil in isoenergetic alfalfa diets was not 0.9 and mean BW of 336 ± 31 kg. The advantageous in minimizing or preventinitial DE intake of 50% of maintenance ing hypophosphatemia or hypowas gradually increased to 100% during magnesemia for refeeding starved horses. the 10-d refeeding period. Two of the The AH diet is recommended as more horses receiving the AH diet were supportive in refeeding starved horses euthanized, thus 12 horses (AH, n = 7; because of the greater intake of dietary AO, n = 5) survived the 10-d study. One phosphorous and magnesium. preprandial and four postprandial venous blood samples were collected daily. (Key Words: Equine, MalnourishDietary effects (P<0.01) were shown for ment, Starvation, Insulin, Phosphoblood serum or plasma free fatty acids rus.) (FFA), phosphate, magnesium, and 2,3diphosphoglycerate (2,3-DPG), but not glucose, insulin, or potassium concentraChronically starved horses are tions. The response of insulin and FFA during the 10-d study showed differences similar to emaciated humans in their response to refeeding; they experience (P<0.05) between AH and AO diets. a broad range of metabolic derangeSerum phosphate concentration in horses ments including severe hypoon both diets exhibited a general slow decline with concentrations below normal magnesemia and hypophosphatemia with a decrease in 2,3reference range during d 5 through 10, diphosphoglycerate (2,3-DPG) concentration, which is important in the 1To whom correspondence should be adhemoglobin-oxygen association in dressed: [email protected] the red blood cell (Witham and Stull,
Introduction
1998). Anecdotal reports suggest that abrupt refeeding of horses may cause death within 3 d (Kronfeld, 1993). Limited reports have evaluated practical diets for refeeding chronically starved horses (Kronfeld, 1993; Finocchio, 1994). Our earlier study on refeeding starved horses showed that isoenergetic diets composed of either alfalfa or oat hay produced lower insulin and glucose responses than an isocaloric diet consisting of a combination of oat hay and a commercial complete feed (Witham and Stull, 1998), a result consistent with clinical experiences (Kronfeld, 1993; Finocchio, 1994). The benefits of corn oil supplementation in equine diets have been well documented over the last decade. One benefit to the starved horse is that the postprandial response of insulin in healthy horses is less with the addition of corn oil, which may be advantageous to lessen hypophosphatemia and hypomagnesemia frequently found with the refeeding syndrome (Stull and Rodiek, 1988). Thus, the objective of this study was to examine the metabolic responses of chronically starved horses during refeeding using two isoenergetic diets consisting of either 100% alfalfa hay (AH) or 80% alfalfa hay with 20% corn oil (AO), on a caloric basis.
48
Materials and Methods
Stull et al.
treatment, and five horses were assigned the AO diet. Horses had free Horses and Study Design. All access to water throughout the study, phases of the study were approved by but no supplemental salt or minerals the University of California Animal were available. Care and Use Committee. Two 10-d Alfalfa hay samples from each trial feeding trials were conducted in 1996 were analyzed by an AOAC-approved and 1999 using 14 mature horses. laboratory (JL Analytical Service, Emaciated horses of mixed light Modesto CA) prior to the start of breeds (8 mares, 5 geldings, and 1 each trial (Table 1). The AO diet was stallion) and unknown histories were formulated with 80% of the calories purchased in Mexicali, Mexico or from alfalfa hay and 20% of the Albuquerque, New Mexico by a calories from corn oil; hence, intake commercial buyer. Horses were was calculated from the known transported for approximately 8 h by composition of these two ingredients trailer to Davis, California. Horses (Table 2). The following equation for ranged in age from 2 to 15 yr (8.3 ± maintenance was used to calculate 4.9 yr), as estimated by examination the daily DE requirement (Mcal/d) for of teeth, and weighed between 265 each horse; “target” BW was estiand 373 kg (336 ± 31 kg) on arrival. mated to be 125% of each horse’s BW Mean height at the withers was 144 ± at the start of the trial: Mcal DE/d = 4.8 cm. Body condition scores (BCS) 1.4 + 0.03 BW (NRC, 1989). ranged from 1 to 4 (1.8 ± 0.9), using All feed was withheld on arrival (d a maximum score of 9 (Henneke et 0), and the first meal was fed on the al., 1983). Horses in each refeeding following morning (d 1) at 0900 h. trial (Trial 1, n = 7; Trial 2, n = 7) were The DE per day gradually increased emaciated but clinically free of overt from 50% divided into six meals disease, as determined by physical offered at 4-h intervals (0900, 1300, examination. 1700, 2100, 0100, and 0500 h) on d 1 The same barn was used for each to 3, to 75% of daily DE divided into trial to house the horses for the 10-d six meals on d 4 to 5. On d 6 to 10, trial. Approximately 40% of the floor 100% of the DE per day was fed in in each concrete stall (6.8 × 3.5 m) three feedings at 8-h intervals (0900, was covered by rubber mats, and no 1700, and 0100 h). Meals were bedding was provided to eliminate the possibility that horses would TABLE 1. Composition of alfalfa consume the bedding. Horses were hay (AH) fed during Trial 1 weighed daily between 1300 and 1400 (1996) and 2 (1999) and alfalfa h using an electric portable scale hay supplemented with corn oil (Model H90-3042; Fairbanks, St. (AO) fed during Trial 2 (1999). Johnsbury, VT). Diet Formulation and Feeding Trial 1 Trial 2 Protocol. Horses were assigned to one of the two following isoenergetic Item AH AH AO diets: 100% alfalfa hay (AH; n = 9) or 80% alfalfa hay and 20% corn oil DE, Mcal/kg 2.28 2.48 3.78 (AO; n = 5 ). Because the availability Moisture, % 11.9 10.5 8.4 of a sufficient number of emaciated CP, % 21.4 20.0 16.0 horses was limited to meet research Crude fiber, % 22.1 24.9 20.0 objectives and schedules, data from Crude fat, % 2.3 3.4 23.4 horses in a previous study (1996) were Ash, % 7.5 7.2 5.8 combined with Trial 2. Feeding Phosphorus, % 0.44 0.24 0.19 protocols, sampling schedules, and Magnesium, % 0.62 0.46 0.37 housing were replicated between Potassium, % 1.8 1.7 1.4 Calcium, % 0.92 0.70 0.56 trials. All horses in Trial 1 were Sodium, % 0.21 0.11 0.09 assigned the AH treatment. In Trial 2, two horses were assigned the AH
individually weighed for each horse, and all orts were collected, weighed, and recorded prior to the next meal. Blood Collection. On the day of arrival, a 14-ga indwelling catheter was placed aseptically in the left jugular vein of each horse. If functional problems or abnormal swelling occurred at the catheter site, a replacement catheter was inserted in the opposite jugular vein several hours prior to the next blood sampling period to avoid any stress responses caused by catheterization procedures. Each day of the 10-d trial, preprandial blood samples (30 mL) were collected at 0845 h, prior to the first meal (0900 h). Horses were fed their assigned meal at 0900 h. Postprandial samples were drawn every hour starting at 1000 h and continuing until 1300 h for a total of four samples. Blood samples to be analyzed for electrolytes, FFA, insulin, and glucose concentrations were immediately placed on ice and allowed to clot. Serum was obtained from these samples and frozen at –56°C. Additionally, blood samples were collected in evacuated tubes containing potassium oxalate and sodium fluoride for the determination of 2,3DPG concentration. One milliliter of this blood was added to 3 mL of cold trichloroacetic acid and centrifuged; the supernatant was then separated and frozen for storage. Selected electrolyte (magnesium, inorganic phosphate, and potassium) concentrations were quantified using an automated spectrophotometry system (Ectachem DT-60; Eastman Kodak Co., Rochester, NY). Free fatty acids and 2,3-DPG concentration were measured by enzymatic colorimetric assays (Procedure 541®; Sigma Diagnostics, St Louis MO and Procedure 337®; Boehringer-Mannheim, Indianapolis, IN, respectively). Glucose concentration was determined by use of an autoanalyzer (YSI 2300 STAT Plus®;Yellow Springs, OH). Insulin concentration was analyzed using a commercially available radioimmunoassay kit (Kit D1804®; Micromedic Systems, Horsham, PA)
Fat Supplementation for Refeeding Starved Horses
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Bacteriologic cultures were positive for Salmonella saint-paul. A second mare (17 yr; BCS, 1.5) developed mild diarrhea on d 5 while exhibiting behavior consistent with increasTrial 1 Trial 2 ing abdominal pain, which became uncontrollable; euthanasia was Item AH AH AO elected on d 8. Necropsy revealed volvulus of the left large colon at the diaphragmatic flexure. It is Offered DE, Mcal/d 6.59 7.31 7.09 unknown if the AH diet contributed Offered feed, kg/d per horse 2.89 2.95 1.87 Consumed feed, kg/d per horse 2.84 2.90 1.78 specifically to the cause of death, as CP, g/d per horse 609 580 285 the number of horses that were Crude fiber, g/d per horse 628 722 355 euthanized was small, and the Crude fat, g/d per horse 65 99 417 causes of death were seemingly Ash, g/d per horse 213 209 103 unrelated. Only data from the 12 Phosphorus, g/d per horse 12.5 7.0 3.4 horses (AH diet, n = 7; AO diet, n = Magnesium, g/d per horse 17.6 13.3 6.6 5) that survived the 10-d study are Potassium, g/d per horse 51.1 49.3 24.2 included in the statistics. Calcium, g/d per horse 26.6 20.3 10.0 Two different sources of alfalfa Sodium, g/d per horse 6.0 3.2 1.6 hay were used for Trials 1 and 2. The nutrient content of each of the two hays (Table 1) was typical for validated for equine blood by Reimers assumptions of normality and high quality alfalfa hay (NRC, 1989). et al. (1982). homoscedasticity were examined A preliminary statistical analysis Statistical Analyses. Repeated prior to finalizing the analysis, using compared the responses of horses on measures ANOVA (PROC GLM; SAS Wilk-Shapario (Wilk and Shapiro, the AH treatment from 1996 and Inst. Inc., Cary, NC) was used to 1968) and Levene tests (Levene, 1960). 1999 to determine whether it was examine the effects of trial, time Variables that required transformaappropriate to combine these groups (daily), and diet on daily blood tion (using logs) were analyzed as in a single analysis. Those results are responses of each horse to the meal logs. Significance is claimed whenshown in Table 3. There were tempooffered at 0900 h. For the parameters ever P<0.05. ral (day-to-day) responses (P<0.05) for of glucose, insulin and FFA, the all variables except 2,3-DPG and magnitude of the postprandial potassium, and trial effects (P<0.05) increment response from the were shown for FFA, 2,3-DPG, magneThe horses used in this study were preprandial (0845 h) measurement sium, and inorganic phosphate. purchased in an emaciated stage of was calculated as the area under the However, because there were no starvation, but were otherwise curve using the trapezoidal method significant time × trial interactions healthy, with no signs of disease or and log-transformed data. For 2,3(P>0.05 for all parameters), indicating lack of dental function. The poor DPG concentration and serum similar responses of the parameters condition of these horses was probelectrolytes, including inorganic during the 10-d study, the data from ably due to economic hardship phosphate, magnesium, and potasthe trials were combined for the AH experienced by the owner or unavail- diet (n = 7). sium, repeated measures ANOVA was based on log-transformed data using able forage during drought condiThe primary aim of this study was a weighted mean. For these variables, tions. The horses were representative to determine the metabolic responses postprandial peaks were not expected; of cases presented to humane officers, of starved horses to two isoenergetic nutritionists, veterinarians, and therefore, these were measured only diets with and without the inclusion rescue shelter personnel for rehabilionce or twice following the meal. of corn oil. As expected, the protein, tation. All horses were ambulatory The Huynh-Feldt adjustment was fiber, and mineral content was less in used with the univariate results as an following transport from the site of the AO diet as compared with the AH purchase. Two of the 14 horses in adjustment for serial correlation of diet (Table 2) because of the nutrithe residual (Huynh and Feldt, 1976). Trial 1 were euthanized prior to the tional dilution caused by the addiend of the 10-d study. A 7-yr-old Post-hoc analyses were conducted tion of corn oil. Because of the desire mare (BCS, 2.5) assigned the AH diet using pairwise contrast tests (for to examine practical equine diets, no refused to eat after the first meal, mean effect of diet) or contrast effort was made to formulate these developed profuse watery diarrhea on diets on an iso-nitrogenous or other decomposition (for time × diet d 2, and was euthanized on d 4. interactions). For all analyses, the nutrient-equivalent basis.
TABLE 2. Calculated daily feed and nutrient intakes of alfalfa hay (AH) during Trial 1 (1996) or Trial 2 (1999) or of alfalfa hay and corn oil (AO) during d 1 to 3 (50% DE/d).
Results and Discussion
50
Stull et al.
pared with other common feeds including both forages and concentrates. There was an approximate twofold difference in CP intake between the diets (Table 2); thus, the amino acid intake also was doubled Item P (Trial) P (Time) P (Time x trial) with the consumption of the AH diet. This increased intake of amino Glucose 0.1698 0.0189 0.1242 acids likely contributed to the greater Insulin 0.1350 0.0009 0.1596 response of insulin from the AH diet Free fatty acids 0.0281 <0.0001 0.2987 during the 10-d study. Interestingly, Phosphate 0.0340 <0.0001 0.3201 arginine-induced insulin release 2,3-diphosphoglycerate 0.0136 0.7397 0.6349 occurred more consistently in mares Magnesium 0.0091 0.0003 0.4401 than in geldings and stallions Potassium 0.1067 0.3278 0.6701 (Sticker et al., 2001). The number of horses in this refeeding study was too limited to examine responses because Feed Consumption and BW Gain. tion was significant (P<0.0001), of gender, but gender may be a A general decline in BW occurred especially as the DE per meal inpotential factor in manipulating between d 1 and 5 when 50 to 75% of creased during d 6 to 10 (Table 4; insulin response through dietary the daily DE requirement was offered; Figure 1). No difference (P=0.17) in mechanisms. small increases in BW were shown insulin response was shown between Concentrations of glucose exhibbetween d 6 and 10 when 100% of diets; however, the interaction of day ited a daily postprandial fluctuation the daily DE requirement was fed and diet (time × diet) was significant within each diet (Figure 1), but there (Figure 1). No difference (P>0.05) in (P=0.03), with the mean insulin was no difference (P=0.98) in the BW response was observed between response of the AH diet exhibiting glucose response between diets over diets during the 10-d study period consistently greater concentrations the 10-d study (time × diet) (Table 4). (Figure 1). The mean BW on arrival than the AO diet over the 10-d study The very low soluble carbohydrate and d 10 was 325 ± 35.6 and 325 ± (Table 4). content of both diets along with the 33.9 kg for horses fed the AH diet and In other studies on fed healthy effect of insulin accounts for the lack 340.7 ± 20.8 and 334 ± 22.1 kg for horses, the roughage-to-concentrate of difference (P=0.99) in serum horses fed the AO diet, respectively. ratio of the diet has affected the glucose response between the diets It was not expected that BW gain insulin response, but mainly because during the 10-d study. On a daily would occur during the 10-d study, of the carbohydrate content of the basis, the maximum glucose concenespecially because only 50 to 75% of concentrate portion (Stull and tration was reached 2 to 3 h followthe daily DE requirement was offered Rodiek, 1988; Powell et al., 2000). ing the morning meal. Mean poston d 1 to 5. The palatability of both The substitution of 20% of dietary prandial glucose concentration did diets by the starved horses was calories from alfalfa hay to corn oil not exceed the reference of 99 ± 0.1 evidenced by a consumption of 98.4 in the AO diet reduced the soluble mg/dL reported in healthy horses fed ± 2.7 and 95.1 ± 4.6% of the offered carbohydrate component of the diet alfalfa diets (Stull and Rodiek, 1988). AH and AO diets, respectively, even and might have contributed to the FFA Concentration. Fats are with the inclusion of a high portion lower insulin response in the AO commonly added to equine diets as a of corn oil (20% of the total calories) treatment. However, the soluble source of energy and contribute in the AO diet. carbohydrate component of alfalfa is about three times more DE than the Insulin and Glucose Response. very low in starch (1.6%) (Anon., same weight of grain (NRC, 1989). The mean preprandial insulin con1971), so the reduction effect in the Usually fat is added at about 5 to centrations in horses fed either diet soluble carbohydrates with the 10% of the total diet, but may be were generally comparable to the substitution of corn oil would be supplemented up to 20% of the diet concentration of 4.7 ± 1.0 µU/mL minimal. on a DM basis. Plant sources of fats previously reported (Stull and Rodiek, Infusion of the amino acids lysine such as corn oil, peanut oil, and 1988). The daily response of insulin and arginine, but not aspartic acid or soybean oil are up to 94% digestible concentration following a meal was glutamic acid, have been shown to (Rich et al., 1981). Corn oil was similar to the pattern of the glucose stimulate the release of insulin in selected for this study because of its response, with peak insulin concenhorses (Sticker et al., 2001). Alfalfa wide availability for purchase by trations generally reaching 3 to 4 h has a comparatively high content of horse owners and acceptable palatfollowing the meal. A temporal both lysine (0.90%) (NRC, 1989) and ability by horses (Holland et al., response (day) of insulin concentraarginine (0.79%) (NRC, 2001) com1993). In an earlier equine study
TABLE 3. Repeated measures of log-transformed blood concentration data of the alfalfa diet for Trial 1 (1996) and Trial 2 (1999) (based on daily means). The reported P values reflect a Huynh-Feldt adjustment for autocorrelation among the daily means.
Fat Supplementation for Refeeding Starved Horses
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Figure 1. Mean BW and concentrations of glucose, insulin, FFA, inorganic phosphorus, 2,3-DPG, magnesium and potassium during d 1 to 3 (50% DE/d fed), d 4 to 5 (75% DE/d fed), and d 6 to 10 (100% DE/d fed) in treatments of alfalfa hay (AH, n = 7) and alfalfa hay and corn oil (AO, n = 5).
52
using healthy horses, the postprandial insulin response was dampened when oil was added to a single meal of corn (Stull and Rodiek, 1988). A reduction in insulin release is proposed to be advantageous in controlling the intracellular shift of phosphorus, which may contribute to a hypophosphatemic state during the initial refeeding period of starved horses. The serum concentration of FFA showed daily responses, dietary differences, and differences between the diets (P<0.001) during the 10-d study (time × diet) (Table 4). The concentration of FFA showed a rapid temporal decrease following the ingestion of the first meal on d 1 (Figure 1). Distinctly high FFA concentrations in preprandial samples were found in the AO treatment throughout the 10-d study. This result is in contrast to the AH group, which displayed elevated preprandial FFA concentrations only during d 1 to 5, with elevated values diminishing and subsequently disappearing during d 6 to 10 when 100% of the daily DE was offered in three meals per day. The range in preprandial FFA concentration in the horses receiving the AO treatment was 0.15 to 0.28 mmol/L during d 6 to 10, with all mean postprandial FFA concentrations measured at <0.15 mmol/L. In contrast, the mean preand postprandial FFA concentrations in horses receiving the AH treatment during d 6 to 10 ranged from 0.02 to 0.07 mmol/L, which is less than the preprandial FFA concentration of 0.25 ± 0.12 mmol/L reported for healthy resting horses (Stull and Rodiek, 1995). The “peak-and-valley” pattern shown in FFA concentration in the AO-fed horses might have been caused by several factors, including rate of digestion and metabolism of nutrients. The total feed offered from the AO diet was 60% less than that offered from the AH diet, with the oil component of the diet known to be highly digestible (Rich et al., 1981). The rapidly available energy from the AO diet apparently reduces
Stull et al.
the need for FFA mobilization from adipose tissue, thereby resulting in a decline in circulating FFA concentrations. However, the readily digestible and absorbed dietary fat is also rapidly cleared (within several hours) from the blood. This relatively rapid appearance and disappearance of dietary fat from the blood dissipates several hours after the previous meal, with little or no dietary fat sources in the preprandial blood sample collected at 0845 h. During this time, the energy needs of the body tissues are likely met by FFA mobilized from stores in the adipose tissue. Thus, the “valley-and-peak” availability of the dietary energy from the AO diet may explain an inverse “peak-and-valley” variation in serum FFA concentrations. Compared with the AO diet, the AH diet contained no rapidly available energy source. Alfalfa hay is low in both fat and soluble carbohydrates (Anon., 1971; NRC, 1989) and is digested largely by microbial fermentation in the large intestine. Slow microbial fermentation results in a lower but more constant release of VFAs into circulation, which is exhibited by much less fluctuation in the serum FFA concentration profile, especially during d 6 to 10. It has been reported that mares fed diets containing only 50% of required energy and protein for 7 d showed reduced plasma glucose and insulin concentrations, while FFA concentration rose throughout the period. When switched to the control diet providing 100% energy
and protein requirements, glucose and insulin concentrations returned to control levels within 1 h postprandially, and FFA declined to control levels within 2 h of consuming the diet (Sticker et al., 1995). This sharp postprandial decrease in FFA is similar to the response of FFA to the initial meal in both the AH and AO diets of the starved horses. Increases in FFA have also been reported in fasting ponies (Beatz and Pearson, 1972; Naylor et al., 1980; Bauer, 1983), swine (Baetz and Mengeling, 1971) and lambs (Cole et al., 1988). Because glycogen stores are among the first energy stores to be depleted in starved animals, the enhanced oxidation of FFA provided in the AO diet may inhibit glucose uptake and glycolysis by skeletal and heart muscle, leaving glucose for energy needs of the brain and other vital tissues. Selected Serum Electrolytes. During refeeding of chronically starved humans, energy supplied by carbohydrates stimulates the release of insulin. Subsequently, there is influx into the cells of glucose, water, phosphorus, potassium, and other electrolytes. Hypophosphatemia results because of the depletion of total body phosphorus during starvation and an influx of phosphorus into the cells during refeeding. Severe hypophosphatemia in humans produces significant reduction in intracellular phosphate metabolites in red blood cells, such as ATP and 2,3-DPG, which limits cell metabolism
TABLE 4. Repeated measures analysis of log-transformed data for the comparison of the AH and AO diets (based on daily means). The reported P values reflect a Huynh-Feldt adjustment for autocorrelation among the daily means. Item
P (Diet)
Glucose Insulin Free fatty acids Phosphate 2,3-diphosphoglycerate Magnesium Potassium
0.9125 0.1745 0.0010 0.0486 0.0003 0.0118 0.0986
P (Time) 0.0006 <0.0001 <0.0001 <0.0001 0.0041 0.0029 0.0847
P (Time x diet) 0.9876 0.0346 0.0006 0.4028 0.2449 0.0825 0.9519
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Fat Supplementation for Refeeding Starved Horses
and survivability. This, in turn, leads to increased hemoglobin and oxygen affinity, followed by the impaired release of oxygen delivery to tissues (Lichtman et al., 1971; Knochel, 1977). Cardiac, respiratory, hematological, and neuromuscular compromise follows (Solomon and Kirby, 1990). Isocaloric diets fed to starved horses showed that serum phosphorous concentration decreased dramatically during the first 5 d of refeeding, predisposing the horses to hypophosphatemia (Witham and Stull, 1998). The response of serum inorganic phosphate, 2,3-DPG, and potassium concentrations during the 10-d study were similar (time × diet; P>0.05; Table 4) between the AH and AO diets. The AO group exhibited consistently greater (P<0.05) serum inorganic phosphate concentrations than the horses fed the AH diet (Figure 1). Phosphate concentration showed daily responses (P<0.0001) to the ingestion of a meal (Table 4). The phosphorous concentration of both diets showed similar responses (time × diet, P=0.40) during the 10-d study, with a general slow decline in mean concentration during the study. Mean phosphorous concentration declined below a normal reference range of 3.1 to 5.6 mg/dL (Carlson and Smith, 1990) on d 3 and 5 in horses fed the AH and AO diets, respectively, indicating the onset of a hypophosphatemic condition. The dietary phosphorous intake of the AH group was two- to threefold greater than the AO group, but no difference was shown (P>0.05) between diets in the response of serum phosphate caused by increased intake. Similarly, whole blood 2,3DPG concentrations in each diet were below normal reference range for healthy horses at the initiation of refeeding and exhibited slight decreases throughout the 10-d study, but no differences (P=0.24) existed between diets over time (Table 4). The concentration of 2,3-DPG showed daily responses (P=0.004) to the ingestion of the meal, and greater mean concentrations of 2,3-DPG were
shown (P=0.0003) consistently in the AH treatment group (Table 4; Figure 1). Mean concentrations at all sampling points in both dietary treatments were below reference resting concentration for healthy horses (2.2 ± 0.2 µmol/mL) (Stull, 1983); values ranged from 0.5 to 1.1 and 1.8 to 1.3 µmol/mL for the AO and AH groups, respectively. A decrease in the production of red blood cell 2,3-DPG has been reported in human refeeding following severe weight loss as a consequence of severe hypophosphatemia (Lichtman et al., 1971; Solomon and Kirby, 1990), and the declining 2,3-DPG concentrations during refeeding in horses appear to support this physiological complication of severe hypophosphatemia. Both diets throughout the 10-d study exceeded the nutrient requirement of potassium for maintenance, which is 20 g/d for a 400-kg horse (NRC, 1989). Serum potassium concentration showed a decrease at the initiation of refeeding in each of the diets, with concentration increasing during the remainder of the 10-d study. The twofold difference in dietary potassium intake between the diets did not affect the response of serum potassium concentration, which was similar (P=0.95) between the diets during the 10-d study (Table 2). Even though potassium concentration was consistently greater in the AO horses, no dietary (P=0.09) or daily effects (P=0.08) were observed (Figure 1). Hypokalemia was not present in horses of either treatment group, as all mean serum potassium concentrations were within the normal reference range (2.4 to 4.7 mmol/L) (Brobst and Parry, 1987). Magnesium Concentration. Hypomagnesemia has been documented as producing synergistic deleterious clinical effects along with hypophosphatemia during the refeeding period in human patients (Holcombe et al., 1985; Vanlandingham et al., 1981). Normal reference range for magnesium concentration in healthy mature horses is 1.3 to 2.5 mg/dL (Brobst and Parry, 1987), and all magnesium
concentrations of the AH horses increased slightly throughout the study (1.3 to 2.3 mg/dL) and remained within the reference range during the study. In contrast, the magnesium concentration of the AO group was either at the low end or below reference range, indicating mild hypomagnesemia. This was reflective of the 2- to 2.5-fold greater daily intake of dietary magnesium (Table 2) in the AH group. An earlier study with starved horses offered a diet of alfalfa hay showed increasing serum magnesium concentrations that were not evident in the subjects fed either oat hay or a combination diet, which had a lower magnesium concentration than the alfalfa diets (Witham and Stull, 1998). Dietary effects (P=0.012) confirmed magnesium concentration was generally less in the AO group compared with the AH group throughout the study (Table 4; Figure 1). The daily effect (P=0.003) of magnesium concentration was shown as the postprandial sample was consistently greater in magnesium concentrations as compared with the preprandial sample, especially during d 6 to 10. The response between diets during the 10d study (time × diet) tended (P=0.08) to be greater in the AH group as compared with the AO treatment. The substitution of corn oil for alfalfa does not appear to be advantageous in minimizing or preventing hypomagnesemia during refeeding.
Implications Hypophosphatemia developed during the 10-d refeeding period for horses in both the AH and AO groups, even though the insulin effect was dampened by the substitution of corn oil in the AO diet. Additionally, mild hypomagnesemia was associated with the AO diet; whereas, the serum magnesium levels in the AH diet increased during the 10-d study. The interrelationship of hypomagnesemia with severe hypophosphatemic condition of the AO diet may produce synergistic deleterious effects; thus, the AH diet
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is recommended as more supportive of the refeeding of the starved horse because it provides a greater intake of dietary phosphorous and magnesium. Nutrient analyses of feeds should be performed in practical settings for the nutritional rehabilitation of starved horses to ascertain the levels of important nutrients, especially phosphorus and magnesium. Future research should examine the effects of supplemental sources of magnesium and phosphorus during the refeeding of starved horses.
Acknowledgments The project was supported by the Center for Equine Health with funds provided by the Oak Tree Racing Association, the State of California Pari-Mutuel Fund, and contributions by private donors. The authors also thank Purina Mills Inc. for additional funding of the research, the pathologists at California Animal Health and Food Safety Laboratory System for performing the necropsy procedures, Neil Willits for statistical assistance, and Kelly Weaver and Marsha Feldman for laboratory analyses. The assistance of Sheila Bruce, Angela Hermann, Kelsie Luiz, Lisa Machado, and Chad Stewart is greatly acknowledged for the care of the horses.
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