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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Department of Animal and Range Sciences, South Dakota State University, Brookings 57007;
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Iowa Department of Agriculture and Land Stewardship, Ankeny 50023; and
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Department of Animal Science, Iowa State University, Ames 50011
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: cattle • feed intake • ghrelin • growth hormone
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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reported that 50% of the herd-to-herd variation in profitability among beef cow/calf operations can be attributed to variation in feed costs. Additionally, inadequate or dramatic fluctuations in feed intake can cause ketosis or acidosis and result in inefficient production of meat (Baile and Della-Fera, 1981). Furthermore, voluntary feed intake often is compromised during stress associated with stage of production and temperature extremes (Baile and Della-Fera, 1981). Understanding the regulation of feed intake in cattle would minimize economic loss and maximize animal well-being in production situations where feed intake, and therefore nutrients available for production, is compromised.
Ghrelin is a peptide hormone synthesized by the abomasal and ruminal tissues of cattle (Hayashida et al., 2001; Gentry et al., 2003). In rodents, ghrelin stimulates feed intake through neuropeptides such as neuropeptide Y (NPY) and agouti-related protein (AGRP) located in areas of the hypothalamus involved in feed intake regulation (Inui, 2001; Nakazato et al., 2001; Shintani et al., 2001). Ghrelin also has been reported to influence energy metabolism in rodents (Tschöp et al., 2000). Whereas research has demonstrated that plasma ghrelin concentrations fluctuate relative to feed intake in sheep (Sugino et al., 2002a,b), it has failed to demonstrate an effect of exogenous ghrelin injection on feed intake (Iqbal et al., 2006).
Because feed intake and energy metabolism contribute greatly to the nutritional status of cattle and the economic viability of livestock operations, the objective of these experiments was to establish the relationship of ghrelin with DMI in beef cattle and to determine the effects of i.v. pulse injections of bovine ghrelin (bGR) on plasma GH, glucose (GLU), insulin (INS), and NEFA concentrations, length of time spent eating, and DMI in cattle.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Experiment 1
Animals and Treatments.
Experiment 1 was conducted at Iowa State University. Four 2-yr-old Simmental x Angus crossbred steers (BW 450 ± 14.3 kg) were used in a crossover design to determine the fluctuation in plasma ghrelin concentrations in the fed and fasted state and to establish the relationship of plasma ghrelin concentrations with plasma GH, INS, GLU, and NEFA concentrations. Steers were acclimated to a climate-controlled facility during an 11-d preexperimental period. Steers were housed individually in a slat-sided crate with a solid-sided feeder and automated waterer attached. Crates were aligned in 2 rows separated by a 2-m alley. Two animals were placed in crates on either side of the alley. Steers were exposed to a 16-h photoperiod beginning at 0600 each day. Temperature and humidity were maintained at a constant 19°C and 89%, respectively.
A finishing diet composed of 83% corn, 5% alfalfa hay, and 12% corn silage (Table 1) was fed throughout the adaptation and sampling periods. During the adaptation period, all steers were offered feed once daily at 0800 and steers were allowed access to feed until 2000 each day. Daily feed allocation was sufficient to result in a minimum of 10% feed refusal when weighed at 2000. Steers were allowed ad libitum access to water throughout the experiment.
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Jugular Catheter Insertion and Blood Sample Collection.
Indwelling jugular catheters were inserted, using a nonsurgical technique, 1 d before the initiation of the sampling period. Serial blood samples were collected from all 4 steers beginning when the feed had been withheld from the FAST group for 22 h and continuing until the feed had been withheld from the FAST group for 48 h. A total of approximately 3% of the blood volume was collected from each steer during the 48-h sampling period. Blood samples were collected at 10-min intervals from 22 to 24 h of fasting and again at 34 to 36 h of fasting. Blood samples collected from 36 to 48 h of fasting were obtained at 15-min intervals. Sugino et al. (2002a, b) demonstrated the greatest fluctuation in plasma ghrelin concentrations for sheep around feeding time; therefore, in the current experiment, blood samples were collected more frequently (10-min intervals) around feeding time to accurately characterize the fluctuation in plasma ghrelin concentrations.
A 7-mL aliquot of blood was collected into a glass tube containing K3 EDTA (12.15 mg supplied by a 15% solution) for collection of plasma, and an 8-mL aliquot was placed in a glass tube containing no anticoagulant for collection of serum. Both tubes were placed on ice and then were centrifuged at 4°C for 20 min at 1,100 x g. A 1.5-mL aliquot of plasma was acidified with 75µL of 1 N HCl to preserve the integrity of the octanoyl moiety of ghrelin. Plasma samples were stored at –20°C for subsequent measurement of ghrelin concentrations. The remaining plasma was separated into 1.0-mL aliquots and stored at –20°C for subsequent analyses of NEFA and INS. Serum samples were aliquoted and frozen at –20°C for subsequent measurement of GLU and GH concentrations.
Analyses of Hormones and Metabolites.
Plasma ghrelin concentrations were measured using duplicate 100-µL plasma samples with a rat ghrelin RIA (Linco Research, St. Charles, MO). This assay is specific for the first 11 AA of ghrelin and the octanoyl moiety and has been validated for bovine plasma (Wertz et al., 2003). An intact octanoyl moiety is believed to be essential for binding of ghrelin to the GH secretagogue receptor (Inui, 2001). Average intraassay CV for the ghrelin assay in Exp. 1 was 10.5%, whereas the interassay CV was 17.0%. The detectable range for the ghrelin assay was from 5 to 200 pg/tube (50 to 2,000 pg per mL of plasma), and average recovery was 96%. Serum GH concentrations were measured in duplicate 100-µL serum samples with an RIA (Trenkle, 1970). Intraassay CV for the GH assay was 4.6%, and interassay CV was 7.3%. The detectable range for the GH assay was 0.15 to 10 ng/tube (1.5 to 100 ng per mL of serum).
Glucose, INS, and NEFA concentrations were measured in samples collected at 1-h intervals. Plasma INS concentrations were determined using duplicate 25-µL aliquots of plasma with a Linco Ultra-Sensitive Human Insulin RIA (Linco Research), but bovine INS was used as the standard, as per instructions of assay manufacturer. Bovine INS standard was validated in comparison with the human INS standard before initiation of the analyses. Minimal and maximal detectable INS was 0.006 to 0.10 ng/tube, respectively (0.24 to 4.0 ng per mL of plasma). Insulin recovery was 83%, and interassay and intraassay CV for the INS assay were 3.5 and 4.2%, respectively. Plasma NEFA concentrations were determined in triplicate plasma aliquots with a colorimetric assay, according to the manufacturer’s procedures (Wako Chemicals USA Inc., Richmond, VA). The detectable range of the NEFA assay was 62.5 to 1,000 µEq/L, with average intra- and interassay CV of 8.6 and 6.4%, respectively. Serum GLU concentrations were assayed in triplicate using a glucose oxidase kit (Sigma-Aldrich, St. Louis, MO). The detectable range for the GLU assay was 25 to 100 mg/dL, and the average intra- and interassay CV were 1.4 and 15.3%, respectively.
Statistical Analyses.
Ghrelin, GH, INS, GLU, and NEFA concentrations were analyzed as repeated measures in time by using the MIXED procedure (SAS Inst. Inc., Cary, NC), with independent errors that accounted for error correlation during the sampling dates. The model included sampling time, treatment (FED vs. FAST), steer, period, and the interaction of sampling time and treatment as independent variables. Differences in least squares means for ghrelin, GH, INS, NEFA, and GLU concentrations at specific sampling times were evaluated by Fisher’s t-test.
Because ghrelin and GH appeared to have pulsatile episodes, the data were divided into 2 sets: prefeeding samples collected at 10-min intervals and postfeeding samples collected at 15-min intervals. Data sets were subjected to the methods of Veldhuis and Johnson (1986) for measurement and characterization of hormone pulses. Mean pulse concentration, number of pulses, average pulse height, and average pulse nadir were assessed for each individual steer within a data set. Pulse amplitude then was calculated as the difference between average peak height and average nadir. These variables were analyzed using the MIXED procedure of SAS. Additionally, the treatment day was divided into 4 periods of 0600 to 0750, 0800 to 1145, 1200 to 1545, and 1600 to 2000. Frequency of pulses within a period was analyzed by using the MIXED procedure of SAS. The model included sampling time, treatment (FED vs. FAST), steer, period, and the interaction of sampling time x treatment as independent variables.
Experiment 2
Verification of Synthesized Ghrelin Activity.
The objective of the second experiment was to determine the effects of i.v. pulse injections of bGR on plasma GH, GLU, INS, and NEFA concentrations and to evaluate effects of exogenous bGR on length of time spent eating and DMI.
Bovine ghrelin was synthesized at the Iowa State University Protein Facility (Ames, IA) using the Fmoc Solid Phase method with a peptide synthesizer (model 432A, Applied Biosystems, Forest City, CA). During synthesis, the hydroxyl group of the third residue (serine) was protected. This protection group was cleaved after synthesis by using 1% trifluoroacetic acid and dichloromethane, and the serine was then acylated with n-octanoic acid in the amino acid synthesizer.
A preliminary experiment using 2 steers fitted with an indwelling jugular catheter was conducted to verify that the synthesized bGR was detectable in blood by using the Linco assay and to verify that bGR was biologically active, as determined by elevated plasma GH concentrations. Blood samples (10 mL) were collected at 15-min intervals from 0600 to 1800 h and processed for the separation of plasma as described for Exp. 1. Two i.v. pulse injections of bGR were given through the jugular catheter at 1300 and 1500. After each injection, the catheter was flushed with 5 mL of saline (SAL). Exogenous bGR (0.08 µg/kg of BW) was injected to achieve a targeted peak concentration in plasma of 1,000 pg/mL. This dosage was chosen based upon results of Exp. 1, in which peak plasma ghrelin concentrations were 1,000 pg/mL for fasted steers. Steer blood volume was estimated to be 8% of BW (Melbin and Detweiler, 1993). Intravenous injection was chosen as the means of delivery to achieve a plasma ghrelin concentration similar to that measured for fasting steers and because feed intake in response to subcutaneous administration in rats was attenuated (Tschöp et al., 2000). Treatment injection times for the preliminary experiment and the complete experiment were selected on the basis of the observation that these steers did not consistently consume feed during this time period. Therefore, steers were classified as being in a satiated state when bGR was administered, and plasma ghrelin concentrations were expected to be at baseline. This model allowed us to determine whether the fasting concentration of ghrelin was adequate to stimulate feeding in a satiated steer. One plasma aliquot was assayed for ghrelin concentration. The ghrelin assay described in Exp. 1 was validated in the laboratory at South Dakota State University and resulted in 94% recovery, 11.8% intraassay CV, and 13.6% interassay CV.
The second aliquot of plasma was frozen at –20°C for subsequent measurement of GH concentration by using the following ovine GH RIA. Plasma concentrations of GH were determined using duplicate 200-µL samples of plasma with materials and procedures provided by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, Bethesda, MD). The following reagents were added to 12 mm x 75 mm polypropylene tubes: 100 µL of assay buffer (0.1% gelatin, 0.9% NaCl, 0.01 M PO4, 0.01% sodium azide, 0.01 M EDTA, 0.05% Tween-20, pH = 7.2), 100 µL of primary antibody [final dilution in the tube of 1:150,000 of NIDDK-anti-oGH-3 (AFP-0802210) in assay buffer], and 200 µL of plasma or a standard solution of NIDDK-oGH-I-5 (AFP-12855B) in assay buffer ranging in mass from 0.15 to 10 ng/tube. Samples and standards were incubated overnight at 4°C.
Labeled tracer, [125I]-ovine GH (NIDDK-oGH-I-5; AFP-12855B), was then added at 25,000 cpm per tube in 100 µL of assay buffer. Samples and standards were incubated overnight at 4°C, then precipitated after a 15 min, 22°C incubation with a preprecipitated sheep-anti-rabbit second antibody. The precipitate was obtained by centrifugation at 1,500 x g, and the supernatant was discarded. Assay tubes containing pellets were counted for 1 min on a gamma counter (Wizard 1470 Automatic Gamma Counter, PerkinElmer, Wellesley, MA). Standards and pooled aliquots of bovine steer plasma were linear (log-logit transformation) and parallel over a mass of 0.15 to 10 ng/tube and a plasma volume of 25 to 250 µL, respectively. The minimal detectable concentration was 0.15 ng/tube. The intraassay CV was 7.8%, and the interassay CV was 18.7%.
Animals and Feed Intake.
After verification that the synthesized exogenous bGR resulted in elevated plasma ghrelin and GH concentrations, 6 steers (BW 416 ± 17.2 kg) were used in a crossover design to determine the effects of i.v. pulse injections of bGR on plasma GH, GLU, INS, and NEFA concentrations, length of time spent eating, and DMI. These 2-yr-old Simmental x Angus crossbred steers were acclimated to a climate-controlled facility and a specific feeding schedule during a 10-d pretreatment adaptation period. Steers were exposed to a 16-h photoperiod, and feed was offered once daily as described for Exp. 1. Steers were fed a common finishing diet throughout the experiment (Table 1
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Each feeding apparatus was attached to a digital load cell that relayed weight differences to a computer (Rice Lake Weighing Systems, Rice Lake, WI). Feeder weight data were logged at 20-s intervals. High variability of logged weights indicated that a steer was eating, whereas a consistently stable weight indicated that the animal was not eating. The difference between a stable weight before and after a highly variable weight period was used to calculate DMI. Each feeding apparatus was hung inside a 3-sided enclosure in which spilled feed could accumulate. During the sampling period, spillage was minimal (
0.10 kg), and therefore differences in weight were assessed as DMI. These data were used to calculate length of time spent eating and DMI, in grams, and DMI per unit of metabolic body size (BW kg0.75). Dry matter intake was recorded 2 d before treatment, the day of treatment, and 1 d after treatment.
Jugular Catheter Insertion and Blood Sample Collection.
The experiment was arranged as a crossover design with 2 treatment periods. Steers were fitted with an indwelling jugular catheter after the adaptation period, and serial blood samples (14 mL) were collected as described for the preliminary experiment. A total of approximately 2% of blood volume was collected from each steer during the sampling period. Blood samples were processed for the separation of plasma and aliquoted, as described for Exp. 1, for the subsequent analyses of ghrelin, GH, INS, GLU, and NEFA concentrations.
During the first treatment period, 3 steers were assigned to the bGR treatment and were given an i.v. injection of bGR through their indwelling catheter at 1200 and 1400. The bGR dosage was calculated as described for the preliminary experiment. After each injection, the catheter was flushed with 5 mL of SAL to ensure that residual bGR from the pulse was rinsed from the catheter. The remaining 3 steers were used as controls and were given 0.9% SAL through the indwelling catheter at a volume equal to that of the bGR bolus and SAL flush. Catheters were removed after collection of the last blood sample, and steers were allowed a 5-d rest between treatment periods. On the final day of the rest period, indwelling jugular catheters were reinserted, bGR and SAL treatments were switched between steer groups, and the treatment period was repeated as described for period 1.
Analyses of Hormones and Metabolites.
Plasma ghrelin and GH concentrations were determined as described for the preliminary experiment. Plasma GLU, INS, and NEFA concentrations were quantified as described for Exp. 1.
Statistical Analyses.
Two steers were removed from the experiment because of catheter malfunction during a treatment period. Therefore, statistical analyses were performed on data from 4 steers that completed both treatment periods. Plasma ghrelin, GH, GLU, INS, and NEFA concentrations were analyzed statistically as repeated measures in time by using the MIXED procedure of SAS, as described for Exp. 1, with the exception that data were not evaluated for pulsatility.
Length of time spent eating and DMI data were divided into 2 data sets. A short-term data set was established to evaluate the acute effects of ghrelin injection on the day of treatment. The long-term data set was used to compare DMI and length of time spent eating on treatment day with that before and after treatment. The objective of these analyses was to determine if the effects of ghrelin treatment on feed intake had an impact on subsequent feeding behavior. Both data sets were analyzed as a crossover design by using the MIXED procedure of SAS. Differences in least squares means for DMI or length of time spent eating due to treatment were evaluated by using PDIFF option of SAS.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). Plasma ghrelin concentrations remained elevated throughout the sampling period and increased for FAST steers as the length of fast progressed. Average plasma ghrelin concentrations from 22 to 24 h of fasting were less (P < 0.001) when compared with plasma ghrelin concentrations from 46 to 48 h of fasting (468 and 802 ± 21.8 pg/mL, respectively). Differences in plasma ghrelin concentrations were not observed for steers in the FED treatment group during the time frame that correlated with 22 to 24 and 46 to 48 h of fasting (86.6 and 76.8 ± 21.8 pg/mL, respectively).
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). Average plasma ghrelin concentration prefeeding and postfeeding was 174 and 102 ± 4.2 pg/mL, respectively. Although plasma ghrelin concentrations were elevated throughout the sampling period for FAST steers compared with those for FED steers, there was no characteristic rise and subsequent fall in plasma ghrelin concentrations that corresponded with the time of feed offering to the FED treatment group (Figure 2). The number of detectable ghrelin pulses for FAST steers from 36 to 48 h of fasting was similar to the number of pulses detected for FED steers during the same time frame. However, average pulse concentration was greater (P < 0.001) for FAST compared with FED steers (820 and 165 ± 53.2 pg/mL, respectively). Average nadir for ghrelin pulses for FED steers was less (P < 0.001) than average nadir for FAST steers (77 and 531 ± 41.2 pg/mL, respectively). Pulse amplitude from average nadir to average pulses height was greater (P < 0.01) for FAST steers compared with FED steers (288 and 88 ± 31.9 pg/mL). There was no difference in ghrelin pulse frequency as a result of treatment; however, pulse frequency was greater from 1200 to 1545 regardless of treatment.
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). As observed for plasma ghrelin concentrations, average serum GH concentrations also were greater (P < 0.05) from 46 to 48 h of fasting compared with 22 to 24 h fasting (3.7 vs. 2.5 ± 0.4 ng/mL, respectively). Differences in serum GH concentrations were not observed for steers in the FED treatment group during the time frame that corresponded to 22 to 24 h and 46 to 48 h of feed deprivation. For the FED treatment group, average serum GH concentrations were elevated (P < 0.001) during the 2-h prefeeding period compared with the average for the remainder of the sampling period (3.9 and 1.9 ± 0.24 ng/mL, respectively). However, serum GH concentrations for FED compared with FAST steers were similar during the 2-h prefeeding period (3.9 and 2.5 ± 0.75 ng/mL), and there was no difference in the number of pulses observed during this time. In contrast, from 36 to 48 h of fasting, a greater number of GH pulses (P < 0.02) and greater (P < 0.05) average pulse GH concentration was observed for FAST compared with FED steers (4.3 and 1.6 ± 0.76 ng/mL, respectively) during the same time frame (Figure 4). An increased (P < 0.05) frequency of GH pulses was detected from 1200 to 1545, regardless of treatment. Increased GH pulse frequency between 1200 and 1545 corresponds to an increased ghrelin pulse frequency during this same time frame. For 3 of the 4 steers in the FED treatment group, plasma GH concentrations were positively correlated (P < 0.02) to plasma ghrelin concentrations. A correlation between GH and ghrelin was not detected for steers in the FAST treatment group.
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). Despite differences in plasma ghrelin and INS concentrations for cattle in the FED and FAST treatment groups, serum GLU concentrations were similar, regardless of nutritional state (Figure 5B). As an indication of nutritional status of these cattle, plasma NEFA concentrations were measured (Figure 5C). Plasma NEFA concentrations were elevated (P < 0.001) for FAST steers compared with FED steers, indicating that FAST steers were in a catabolic state during the sampling period.
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). Ghrelin was injected to achieve a peak concentration of 1,000 pg/mL, which was the physiological concentration of the pulses detected for fasting steers in Exp. 1. At first postinjection blood collection, 15 min subsequent to bGR injection, mean plasma ghrelin concentrations were 434 ± 50.5 pg/mL. Plasma GH concentration increased (P < 0.005) in response to the first bGR injection (Figure 6B). The second i.v. injection of bGR, given 2 h subsequent to the first, did not result in a significant increase in plasma GH concentrations compared with SAL steers. In this experiment, plasma GLU, INS, and NEFA concentrations were not different for bGR steers compared with those of SAL steers (Figure 7).
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). Length of time spent eating and DMI were similar for treatment groups before bGR injection on the day of treatment. At each of the 1-h postinjection periods, length of time spent eating and DMI were not different between bGR and SAL treatment groups. However, when data for the two 1-h postinjection periods were combined, length of time spent eating was greater (P = 0.02) for bGR steers compared with SAL steers. Additionally, there was a tendency for DMI (g; P = 0.06) and DMI (g/kg of BW0.75; P = 0.07) to be increased for bGR steers during the combined injection times.
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). At each of the measured time points, length of time spent eating and DMI on treatment day were not different from data recorded before treatment or subsequent to treatment.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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; Miura et al., 2004) and sheep (Sugino et al., 2002a,b) support an increase in plasma ghrelin concentrations relative to feeding time. In the current experiment, prefeeding plasma ghrelin concentrations were elevated relative to postfeeding concentrations. However, differences between prefeeding and post-feeding ghrelin concentrations were not as pronounced as those reported by other researchers studying cattle and sheep (Sugino et al., 2002a,b; Miura et al., 2004). Several factors including body size or body composition, feeding frequency, and diet composition or amount offered may contribute to differences in the responsiveness of ghrelin between these experiments. Miura et al. (2004) demonstrated a difference in pre-and postfeeding plasma ghrelin concentrations of approximately 90 pg in mature cows and much less variation in young calves. The cattle in the current experiment were of intermediate size compared with the calves and cows used by Miura et al. (2004), and size or age differences may contribute to the less pronounced increase in plasma ghrelin concentration at feeding. Body composition in addition to body size should be considered in discussion of the responsiveness of ghrelin to feeding. Wertz et al. (2003) demonstrated increased plasma ghrelin concentrations, for steers, as the number of days a steer was fed increased (0 to 196 d). Although not reported in the abstract, ultrasound measurements indicated that subcutaneous fat thickness also increased for these steers as the length of time a steer was fed increased. Kurose et al. (2005) demonstrated greater plasma ghrelin concentrations for sheep with excessive body fat compared with lean sheep. However, the expression of the GH-secreteagogue receptor in the arcuate nucleus was greater for lean sheep compared with sheep with normal or excessive body fat (Kurose et al., 2005). These authors concluded that although plasma ghrelin concentrations are elevated as body fat is increased, fewer receptors for the hormone exist in the area of the hypothalamus that is known to be involved in the regulation of feed intake. Additionally, because more pronounced differences in plasma ghrelin concentrations resulted with multiple feed offerings, feeding frequency should be considered. Sugino et al. (2002b) reported less fluctuation in plasma ghrelin concentrations for sheep allowed ad libitum consumption compared with those offered feed 2 to 4 times daily. Likewise, Miura et al. (2004) offered feed twice daily and reported a more pronounced difference in plasma ghrelin concentration. In the current experiment, steers were offered feed once daily and allowed to consume throughout a 12h period. The amount of feed offered was sufficient to result in at least 10% feed refusal at the end of the feeding period. This design is more similar to ad libitum consumption than offering a meal 2 to 4 times daily and therefore may have resulted in a less pronounced increase in plasma ghrelin concentration prefeeding. Diet composition and amount of feed consumed also may have contributed a less pronounced increase in plasma ghrelin prefeeding. Miura et al. (2004) reported more a pronounced elevation in prefeeding ghrelin concentration for mature cows fed a diet composed solely of forage (intake 1.3% of BW). In the current experiment, intake was 2.3% of BW, and the diet was composed of 80% grain. Therefore, total caloric intake relative to body size was different between the 2 experiments. For calves fed a diet composed of grain and forage at 3.5% of BW, plasma ghrelin concentration fluctuated to a lesser extent relative to feeding. These data are consistent with the hypothesis that feeding frequency, composition and quantity of the diet fed, and animal size or age may influence the responsiveness of ghrelin.
Although fluctuation in plasma ghrelin concentrations relative to feed consumption has been reported for cattle fed daily, no data have been reported that observe plasma ghrelin concentrations for cattle completely deprived of feed. However, for sheep offered feed enclosed in a nylon envelope from which they could not consume (sham-fed), plasma ghrelin concentrations remain elevated, whereas plasma ghrelin concentrations diminished for contemporaries that were allowed to consume the offered feed (Sugino et al., 2002a). Sugino et al. (2002a) concluded that plasma ghrelin concentrations are not diminished simply as a conditioned response to offering feed. Research with rodents (Lee et al., 2002) and sheep (Sugino et al., 2003) has demonstrated that vagal cholinergic neurons exert a constant inhibitory influence on ghrelin-secreting cells. However, the mechanism by which inhibition is alleviated by feed deprivation has not been elucidated. Salfen et al. (2003) demonstrated that plasma ghrelin concentrations remained elevated in feed-deprived pigs through 48 h of feed deprivation and then diminished to concentrations less than those observed for pigs that had access to feed. Because plasma samples were not collected beyond 48 h of fasting in this experiment, it cannot be determined whether plasma ghrelin concentrations for fasted cattle would remain elevated or decline. The differing nature of the monogastric and ruminant digestive anatomy, however, may contribute to differences in plasma ghrelin concentrations during feed deprivation. Additional research is needed to determine the effects of long-term fasting on plasma ghrelin concentrations in ruminants.
Growth Hormone.
Ghrelin is an endogenous ligand for the GH secretagogue receptor and, in rodents, results in the secretion of GH from the anterior pituitary gland that is independent of the GH-releasing hormone axis (Arvat et al., 2001; Inui, 2001). Because divergent plasma ghrelin concentrations resulted for FED and FAST steers, serum GH was evaluated to determine whether differences in mean concentration or concentration pattern resulted between the treatment groups. Elevated serum GH concentrations observed for cattle exposed to an acute feed deprivation (48 h) in the current experiment were similar to those reported for sheep treated to chronic (20 d) undernourishment (Thomas et al., 1991). An increase in the number of GH pulses was observed for steers subjected to acute feed deprivation in the current experiment; however, Thomas et al. (1991) did not observe an increase in the number of GH pulses with chronic undernourishment of sheep. This discrepancy could be the result of differences in severity or length of nutrient restriction. As demonstrated by various researchers, there are multiple means by which serum GH concentrations are increased during undernourishment. Trenkle (1976) reported less turnover of GH pools but no difference in secretion rates for fasting compared with fed calves, suggesting that small increases in serum GH during fasting partially are the result of decreased metabolic clearance. In contrast, Henry et al. (2001) concluded that increased plasma GH concentrations that resulted from chronic undernourishment were the result of increased GHRH and decreased somatostatin synthesis in the hypothalamus. Ghrelin, because it is elevated during fasting and results in increased GH concentrations, also should be considered as contributing to GH secretion during undernourishment. Steers in the FED treatment group exhibited elevated GH during feeding, which was concomitant to the elevation in plasma ghrelin concentration. This feeding-time surge in serum GH has been reported by several researchers for cattle and sheep (Trenkle, 1978; McMahon et al., 2000; Sugino et al., 2002b). Sugino et al. (2002a) observed a characteristic increase of plasma GH for fed sheep and those offered feed but not allowed to consume.
Insulin, Glucose, and NEFA.
Glucose, INS, and NEFA data are consistent with those reported in the literature for cattle in the fed or fasted state (McAtee and Trenkle, 1971; Trenkle, 1976; Rule et al., 1985). In response to fasting, Chelikani et al. (2004) demonstrated plasma GLU, INS, and NEFA concentrations for nonlactating pregnant cows similar to those observed for mature beef steers in the current experiment. However, Chelikani et al. (2004) demonstrated that responsiveness of plasma GLU, INS, and NEFA concentrations during fasting differed depending on physiological state of the animal. Plasma GLU and INS were more responsive to fasting in early-lactation cows and postpubertal heifers than in nonlactating cows.
It has been demonstrated that refeeding or the ingestion of dextrose resulted in decreased ghrelin concentrations in rodents that were fasted (Tschöp et al., 2000); however, ingestion of water and therefore simply stomach expansion did not decrease plasma ghrelin concentrations elevated by fasting. Horvath et al. (2001) reported that the ingestion of carbohydrates decreased plasma ghrelin concentrations elevated by fasting and alluded to a rise in plasma GLU concentrations as a means of regulating plasma ghrelin concentrations. In humans, however, parenteral administration of GLU or INS did not suppress plasma ghrelin concentration (Caixas et al., 2002). It is therefore likely that species differences exist. In ruminants, circulating blood GLU concentrations are approximately 50% that of a monogastric animal (Fahey and Berger, 1988) and GLU concentrations fluctuate to a lesser extent relative to feed consumption (Forbes, 1995). In the fed state, ruminants derive the majority of their GLU from metabolism of propionate in the liver, and therefore less fluctuation in plasma GLU concentrations occurs relative to meal consumption. However, as propionate substrate for gluconeogensis decreases with fasting, GLU sparing is initiated to maintain homeostatic GLU concentrations (Blum et al., 1981; Rule et al., 1985). Increased lipolysis is supported by a increased ratio of GH: INS (Athanasiou and Phillips, 1978; Rule et al., 1985). Mobilization of adipose tissue during fasting supplies NEFA that can be used by body tissue for energy instead of GLU. Additionally, Lyle et al. (1984) demonstrated increased capacity for hepatic gluconeogenesis from alanine, lactate and glycerol, and McDowell (1983) reported decreased uptake of GLU by peripheral cells as a result of decreased INS concentrations. Because multiple mechanisms exist to maintain homeostatic plasma GLU concentrations, it is unlikely that fluctuation of plasma GLU concentrations in ruminants results in a decrease in plasma ghrelin concentrations. However, McCowen et al. (2002) demonstrated a positive correlation between plasma ghrelin concentrations and plasma NEFA concentrations in rodents.
Rats allowed ad libitum consumption of a high-fat diet have been reported to have decreased plasma ghrelin concentrations compared with rats allowed ad libitum access to a mixed-nutrient commercial rat chow (Lee et al., 2002). Because rats were allowed ad libitum consumption, it is plausible that caloric intake and not a given macronutrient mediates plasma ghrelin concentrations. Further research, however, demonstrated that when caloric intake was similar but the source of calories was fat or carbohydrate, either macronutrient decreased gastric ghrelin mRNA (Sanchez et al., 2004). However, upon subsequent fasting, gastric ghrelin mRNA increased for rats assigned to the fat diet but not the carbohydrate diet. Data from rodent experiments suggest that the amount of dietary carbohydrates and therefore plasma GLU concentrations influence fluctuation in plasma ghrelin concentrations associated with feeding in monogastric animals. Because the metabolism of GLU and therefore plasma GLU concentrations fluctuate little in ruminants despite differences in ghrelin concentrations, alternate regulatory mechanisms need to be investigated for ruminant animals.
On the basis of data from this experiment, plasma ghrelin concentrations fluctuate in cattle relative to their nutritional status. Fluctuating plasma ghrelin concentrations are associated with differing GH, INS, and NEFA concentrations; however, plasma GLU concentrations did not fluctuate relative to nutritional status. Whereas dramatic differences in plasma ghrelin concentrations result for feed-deprived cattle compared with those in cattle with adequate nutrition, it has not been addressed whether gradient ghrelin concentrations result with intermediate levels of nutrient restriction. Additionally, ghrelin concentrations have not been evaluated for cattle when intake is limited by physical fill of a high roughage diet compared with a chemical signal of a high-grain diet.
Experiment 2
Ghrelin
Plasma ghrelin concentration expected at the first postinjection sampling (15 min postinjection) was 500 pg/mL; however, the slightly lower ghrelin concentration (434 ± 50.5 pg/mL) measured at the first postinjection sampling may have been the result of metabolic clearance that is closer to 10 min than 15 min. The half-life of ghrelin has been reported to vary among species: humans, 10 min (Nagaya et al., 2001); pigs, 15 min (Salfen et al., 2004); and rats, 30 min (Tolle et al., 2002). Because plasma ghrelin concentrations were only slightly less than expected at the first postinjection sampling period, the authors believe that the target ghrelin concentration of 1,000 pg/mL was achieved. Plasma ghrelin concentrations were elevated (P < 0.001) immediately following each injection but returned to a concentration similar to baseline concentration at 30 min postinjection.
Growth Hormone
These results are consistent with previous data where plasma GH concentrations were elevated during feed restriction. These results may be, in part, the result of elevated ghrelin concentrations and not only the result of decreased metabolic clearance (Trenkle, 1976) or increased GHRH and decreased somatostatin (Thomas et al., 1991; Henry et al., 2001). In the present experiment, bGR was injected to achieve a plasma ghrelin concentration similar to the physiological concentration measured in feed-deprived steers. When expressed on a BW basis, the dosage of bGR (0.08 µg/kg of BW) that resulted in elevated GH concentrations was considerably less than that used in other experiments with cattle (1.0 µg/kg of BW; Itoh et al., 2005). Itoh et al. (2005) reported plasma GH concentrations that remained elevated for 30 to 45 min following i.v. injection of ghrelin. These data are in agreement with GH data reported from the current experiment. Itoh et al. (2005) also reported that plasma GH concentrations in response to ghrelin injection varied depending on age and physiological state of the cattle. Young calves were more responsive than mature cows, and cows in early to midlactation were more responsive than nonlactating cows (Itoh et al., 2005). Plasma GH concentrations in response to ghrelin appeared greater for cattle where nutrient demand is greater (young calves and early to midlactation cows). For mature nonlactating cows, GH concentration in response to ghrelin injection was similar to that of the mature steers in the current experiment, despite differences in injected ghrelin dosage.
In response to the second ghrelin injection, GH was not significantly different from GH concentrations of steers injected with SAL. We conclude from this observation that the GH response to multiple ghrelin injections is attenuated. This conclusion is supported by the data of Salfen et al. (2004) where plasma ghrelin concentrations remained elevated throughout a 5-d ghrelin infusion period, but GH concentrations were not different from SAL-infused pigs at the end of the infusion period. Additional research is needed to establish the effects of sustained elevation of ghrelin on GH concentrations in cattle and the impact of such on rate of gain and body composition.
Insulin, Glucose, and NEFA
Plasma ghrelin concentrations have been demonstrated to acutely decrease with the i.v. infusion of dextrose (rats; McCowen et al., 2002), the ingestion of dextrose (rats; Tschöp et al., 2000; humans; Shiiya et al., 2002), as well as ingestion of a complete meal as demonstrated in the first experiment. In contrast, the responsiveness of plasma GLU and INS to i.v. administration of ghrelin has been mixed. Salfen et al. (2004) reported no acute differences in plasma GLU concentrations as a result of ghrelin injection in pigs. Increased plasma GLU concentrations, however, have been reported for fasted humans injected with ghrelin (Broglio et al., 2001). Differences in the latter 2 experiments are consistent with the hypothesis that the responsiveness of ghrelin varies depending on nutritional state at the time of injection or species differences. Species differences must be considered when comparing the relationship of ghrelin and GLU between ruminant species (cattle) and monogastric species such as pigs, rodents, and humans. As observed in Exp. 1, plasma GLU concentrations fluctuate minimally relative to nutritional status of ruminants. In contrast, plasma NEFA and INS concentrations are responsive to changes in nutritional status of ruminants as demonstrated by Exp. 1. Plasma GLU concentrations in the current experiment did not differ as a result i.v. bGR injection. Previous research indicates that plasma GLU concentrations in response to i.v. ghrelin injection were elevated for lactating cows but less responsive for nonlactating cows and suckling calves (Itoh et al., 2006). As discussed for Exp. 1, cattle in negative energy balance have enhanced hepatic gluconeogenesis and decreased GLU uptake by peripheral tissues to spare GLU for vital functions (McDowell, 1983; Lyle et al., 1984; Bauman, 1999). The lack of responsiveness of plasma GLU to bGR injection in the current experiment may be the result of physiological state and therefore GLU demand, with steers being more similar to the nonlactating cow than the lactating cow. Additionally, the lower concentration of bGR injected in the current experiment compared with that injected by Itoh et al. (2006) also may have resulted in less responsive plasma GLU.
For rats (Lee et al., 2002), pigs (Salfen et al., 2004), and cattle (Itoh et al., 2006), i.v. injection of ghrelin resulted in an increase in plasma INS concentrations. In the current experiment, no differences in plasma INS was observed with bGR injection. When comparing the 3 experiments, it must be considered that species differences or dosage of injected ghrelin contributes to the differing results. The dosage of ghrelin used in this experiment with cattle was 0.08 µg/kg of BW, whereas ghrelin dosages used in previously reported research with cattle (1.0 µg/kg of BW), pigs (2 µg/kg of BW) and rats (720 µg/kg of BW) were considerably greater. McCowen et al. (2002) reported that i.v. INS infusion in rats was sufficient to decrease plasma ghrelin concentrations, but when INS concentrations exceeded physiological range, plasma ghrelin concentrations plateaued. These authors concluded that plasma INS concentrations did not directly influence plasma ghrelin concentrations.
Feed Intake
Ghrelin has been reported to increase feed intake and BW gain (Nakazato et al., 2001; Wern et al., 2001a,b) and to alter body composition in many species including rodents and humans (Tschöp et al., 2000; Wern et al., 2001b). The effects of ghrelin on feed intake reportedly are at the level of the central nervous system and are the result of increased orexigenic peptides, NPY and AGRP, in response to intrace-rebroventricular (ICV) administration of ghrelin (Nakazato et al., 2001; Wern et al., 2001b). However, increased feed intake, BW gain, and altered body composition also have resulted with intravenous, intraperitoneal, and subcutaneous injections of ghrelin (Tschöp et al., 2000; Wern et al., 2001a,b). These data are consistent with the hypothesis that ghrelin can cross the blood-brain barrier and serve as a peripheral signal for central mediation of such factors as feed intake, BW, or body composition (Inui, 2001).
Data from Exp. 1 demonstrated that plasma ghrelin concentrations fluctuate relative to nutritional status in cattle, and these data are supported by previous reports of Hayashida et al. (2001) and Miura et al. (2004). However, in cattle, it has not been demonstrated that exogenous administration of ghrelin can stimulate feed intake. Several factors, however, should be considered when interpreting these data. Firstly, results are based on a limited number of observations; this likely contributed to a lack of statistical significance despite large numeric differences. Secondly, by design, the concentration of ghrelin injected was based on the concentration measured in feed-deprived steers (0.08 µg/kg of BW); however, bGR was injected into steers in a satiated state. Therefore, it is possible that the concentration of circulating ghrelin that would stimulate a feed-deprived steer to consume feed is not sufficient to overcome satiety factors and stimulate satiated cattle to consume feed.
In other experiments where peripheral ghrelin administration has resulted in increase in an feed intake in rodents, injected ghrelin concentrations were considerably greater (Tschöp et al., 2000; Wern et al., 2001b). Additionally, those experiments involved multiple injections over several days. Salfen et al. (2004) reported that multiple ghrelin injections over a 6-d period resulted in increased BW in weanling pigs but no significant increase in feed intake. Likewise, Iqbal et al. (2006) demonstrated that neither ICV injection of ovine ghrelin nor i.v. injection of human ghrelin in sheep influenced feed intake. These researchers concluded that ghrelin does not influence feed intake in ruminant species. However, Salfen et al. (2004) acknowledged when using human ghrelin in a pig model that differences in growth without significant differences in feed intake may have been the result of using cross-species ghrelin. Salfen et al. (2004) speculate that, although cross-species ghrelin sources may stimulate GH, species-specific ghrelin may be necessary to elicit influence on feed intake. In support of their speculation that the effects of ghrelin on feed intake and growth are mediated by different receptors, Nakazato et al. (2001) and Shintani et al. (2001) demonstrated the essential involvement of NPY and AGRP in ghrelin-induced regulation of feed intake. Yet, Tschöp et al. (2000) reported that the NPY cascade was not essential to ghrelin-induced fat accretion because fat accretion was increased similarly in wild-type and NPY-deficient rodents injected with ghrelin. This finding was substantiated by the report that GH secretion, but not feed intake, is stimulated in ghrelin-treated rats in which the arcuate nucleus has been ablated (Tamura et al., 2002). The arcuate nucleus is the site of NPY and AGRP secretion and is considered to be an area of the hypothalamus that is involved in the regulation of feed intake (Tschöp et al., 2000; Inui, 2001). Additionally, Tschöp et al. (2000) reported that feed intake was increased in GH-deficient rats, suggesting that increased feed intake is not the result of a GH surge that has been reported at feeding time. Recently, data indicate that des-acyl ghrelin (octanoyl moiety absent) was not as efficacious as octanoylated ghrelin in stimulating feed intake when administered ICV or i.v. to rats (Toshinai et al., 2006). Des-acyl ovine ghrelin was administered ICV to sheep and did not alter feed intake (Iqbal et al., 2006). These researchers also demonstrated no effect of octanoylated human ghrelin on feed intake in sheep (Iqbal et al., 2006). These data further substantiate the speculation that octanoylated species-specific ghrelin may be necessary to effect feed intake but not GH. Additional research is needed in the area of ghrelin species-specificity and its impact in stimulating GH and feed intake in ruminant species.
In the current experiment, octanoylated bovine-specific ghrelin was used and therefore may account for differences in DMI. Also, it should be considered that 2 i.v. ghrelin injections were given in close proximity and feeding behavior was altered only for the combined treatment period, and not following individual injections. These data are consistent with the hypothesis that multiple i.v. ghrelin injections might be necessary to elicit a feeding response. In contrast to these data, Iqbal et al. (2006) reported that multiple ICV injections of human ghrelin (2.3 µg/kg of BW) did not influence feed intake in sheep. However, because the current experiment with cattle and that reported by Iqbal et al. (2006) are based on a small sample size, additional research is needed to further establish the effects of ghrelin on feed intake in ruminants.
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2 This project was supported by National Research Initiative Competitive Research Grant no. 2004-35206-14372 from the USDA Cooperative State Research, Education, and Extension Service and the Wise and Helen Burroughs Research Endowment.
3 Corresponding author: aimee.wertz{at}sdstate.edu
Received for publication January 30, 2006. Accepted for publication July 20, 2006.
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