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Journal of Animal Science - Animal Physiology

Impact of visual, olfactory, and auditory cues on circulating concentrations of ghrelin in wethers1

 

This article in JAS

  1. Vol. 93 No. 8, p. 3886-3890
     
    Received: Feb 18, 2015
    Accepted: June 03, 2015
    Published: July 24, 2015


    2 Corresponding author(s): jadaniel@berry.edu
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doi:10.2527/jas.2015-9026
  1. M. G. Stockwell-Goering*,
  2. E. A. Benavides,
  3. D. H. Keisler and
  4. J. A. Daniel 2*
  1. * Department of Animal Science, Berry College, Mount Berry, GA 30149
     Division of Animal Sciences, University of Missouri–Columbia, Columbia 65211

Abstract

Ghrelin is a hormone that stimulates feed intake and regulates energy homeostasis. A link has been observed in sheep, in which simulated feedings at scheduled meal times resulted in an increase in ghrelin concentrations. The present study sought to characterize the effect of feeding cues outside of scheduled meal times on circulating ghrelin concentrations in sheep. Katahdin wethers (age 201 ± 4.9 d; weight 35 ± 1.2 kg) were not offered feed (CONT; n = 5), offered 275 g of feed (FED; n = 5), or fitted with a muzzle and offered 275 g of feed (SHAM; n = 5) during the sampling period, which began 2.5 h after normally scheduled daily feeding time. Blood samples were collected via jugular catheter every 15 min for 2.5 h. Feed was offered for 15 min 0.5 h after the start of blood sampling. The CONT samples were collected on d 1, and FED and SHAM samples were collected on d 2. The active ghrelin present in the plasma was then analyzed by RIA. After the Shapiro-Wilk W goodness of fit test demonstrated that 1 SHAM wether was an outlier and it was removed, data were tested for effect of treatment (FED, SHAM, or CONT), time, and treatment × time interaction using procedures for repeated measures with JMP Software (SAS Inst. Inc., Cary, NC). There was no treatment or time effect (P > 0.05); however, there was a treatment × time interaction on plasma ghrelin concentrations (P = 0.0028) such that ghrelin concentrations in SHAM wethers were greater than in CONT wethers 15, 60, and 90 min after feeding, whereas ghrelin concentrations in SHAM wethers were greater than those in FED wethers 30, 60, 90, and 120 min after feeding (P < 0.05). Within the SHAM treatment, ghrelin concentrations were greater at 15 min than at −30 min. Moreover, ghrelin concentrations within the FED treatment were greater at −30 min than at 30, 45, 60, 90, 105, and 120 min and at −15 min than at 15 through 120 min. The area under the curve representing circulating concentrations of ghrelin in CONT, FED, and SHAM treatments, determined using the trapezoidal method, yielded a treatment effect with a tendency toward significance (P = 0.0866). These results indicate plasma ghrelin concentrations in scheduled meal-fed wethers are elevated following visual, olfactory, and auditory feeding cues outside of scheduled feeding times.



INTRODUCTION

Ghrelin, an orexigenic hormone, is the endogenous ligand for growth hormone secretagogue receptor subtype 1a (GHS-R1a; Kojima et al., 1999). Since it was first isolated, ghrelin has been identified in several species, including sheep (Kojima et al., 1999; Kaiya et al., 2003). In ruminants, the primary site of production for secretion is the abomasum (Hayashida et al., 2001; Huang et al., 2006). However, ghrelin’s Ser-3 hydroxyl group must be acylated with n-octanoic acid to bind to GHS-R1a and carry out its functions, which include stimulating GH secretion, feed intake, and adiposity (Tschöp et al., 2000; Grouselle et al., 2008). Such actions are predominantly mediated by the arcuate nucleus in the hypothalamus (Nakazato et al., 2001).

Several studies have sought to elucidate ghrelin’s role. Peripheral ghrelin injections increase circulating concentrations of GH and feed consumption in cattle and ewes (Wertz-Lutz et al., 2006; Grouselle et al., 2008). It was also found that sheep accustomed to scheduled meals experience a preprandial surge of ghrelin (Sugino et al., 2002a). However, the slope of ghrelin’s rise is inversely related to the number of scheduled meals fed and is absent in ad libitum–fed sheep (Sugino et al., 2002b). In his experiments with dogs, Pavlov clearly demonstrated feeding cues (a bell) stimulate digestive tract secretion. Although sheep can discriminate between pictures of full and empty feed buckets (Kendrick et al., 1996), the impact of feeding cues on peripheral ghrelin concentrations in sheep at a time other than that at which a meal is expected has not been studied.

Therefore, given that animals respond to feeding cues, ghrelin stimulates feed intake, and ghrelin can be altered by feeding, this study tested the hypothesis that plasma ghrelin concentrations in scheduled meal–fed wethers would be increased by visual, olfactory, and auditory feeding cues outside of scheduled feeding times.


METHODS AND MATERIALS

Before the experiment, all animal procedures were approved by the Institutional Animal Care and Use Committee of Berry College.

Animals and Procedure

Beginning 14 d before the study, 10 Katahdin wethers (35 ± 1.2 kg, aged 201 ± 4.9 d) were adjusted to daily turnout on pasture at 0800 h. At 1600 h, the wethers were placed on a drylot with free choice hay and group fed 11 kg of pelleted feed (21% canola meal, 20.55% ground barley grain, 20.5% peanut hulls, 20% wheat middlings, 7.5% cottonseed hulls, 6.25% soybean hulls, 2.5% premium lamb base mix, 1.2% calcium carbonate, and 0.5% ammonium chloride), or 1,100 g each. This feeding schedule was continued throughout the study to ensure that ghrelin concentrations declined before sampling began at 1830 h and was designed in compliance with prior research conducted by Sugino et al. (2002a) to increase the uniformity of circulating ghrelin concentrations at the beginning of each sampling period. It also reduced potential variation in ghrelin concentrations among the treatments due to preexisting plasma ghrelin variations or lack of feed availability. Wethers were maintained in the drylot until turnout at 0800 h the next morning. Additionally, all animals had access to water ad libitum throughout the study, and none of the participating wethers were fasted before the meal at 1600 h. All procedures took place in September, at a latitude of 34°18′4″N and longitude of 85°11′40″W, and there was a light-dark period of approximately 12 h. At 1700 h, the day preceding the experiment, all wethers were fitted with indwelling jugular cannulas. They were then randomly assigned to 3 different treatment groups. At 1700 h on the following day, wethers not offered feed during the sampling period, constituting a control group (CONT; n = 5), were placed in individual pens measuring 1.7 m2 from which they could see one another. Beginning at 1830 h, 5-mL blood samples were collected every 15 min for 2.5 h. On d 2, wethers exposed to the sight, smell, and sound of feed as it is placed in buckets but not allowed to eat (SHAM; n = 5) and wethers exposed to feed and allowed to eat (FED; n = 5) were placed in individual pens at 1700 h. Muzzles were placed on SHAM wethers to prevent feed consumption in the presence of visual, olfactory, and auditory feeding cues. Beginning at 1830 h, 5-mL blood samples were collected every 15 min at −30 min, −15 min, and 0 min. Following the sample drawn at 0 min, or 1900 h, all wethers received 275 g of feed. By 1915 h, the FED wethers had consumed their entire allotment and uneaten feed was removed the pens of the SHAM wethers. The sampling period continued until 2100, lasting a total of 2.5 h.

Plasma Hormone Analysis

Immediately after collection, blood samples were placed in tubes spray-dried with 10.8 mg K2-EDTA to prevent coagulation. The plasma was harvested by centrifugation at 1,500 × g at 4°C for 10 min. Following the ghrelin assay manufacturer recommendations (EMD Millipore, Billerica, MA), 50 μL of 1 N HCl and 10 μL of phenomethylsulfonyl fluoride (10 mg/mL in isopropanol; PMSF) were added to 1,000 μL of plasma. Because HCl lowers the pH and PMSF inhibits serine esterases and proteases, the n-octanoic acid bound to the third serine residue of ghrelin was maintained. (De Vriese et al., 2004; Hosoda et al., 2004). The samples were stored at −20°C and shipped to the University of Missouri. Once there, the active ghrelin present in the plasma was measured with a commercially available ghrelin RIA kit (Active Ghrelin Kit GHRA-88HK, EMD Millipore) that was previously validated for sheep by Relling et al. (2009). The minimum assay detectability was 1.66 pg/tube, and the coefficient of variation for both the intra-assay and interassay was <10%. Plasma ghrelin concentrations were expressed as picograms per milliliter.

Statistical Analysis

Ghrelin concentrations were tested for normal distribution using the Shapiro-Wilk W goodness of fit test with JMP software (version 10; SAS Inst. Inc., Cary, NC). One SHAM wether proved to be an outlier and was excluded from all calculations. Using procedures for repeated measures with JMP software (version 10; SAS Inst. Inc.), data were analyzed for effect of group (CONT, FED, and SHAM), time, and group by time interaction. The area under the curve (AUC) representing circulating concentrations of ghrelin in CONT, FED, and SHAM groups was determined using the trapezoidal method. The pre- and postfeeding AUC were then tested for effect of group using standard least squares analysis with JMP software. Means separation was performed using Student’s t test if the main effect or interaction was P ≤ 0.05.


RESULTS

There was a treatment by time interaction (Fig. 1; P = 0.0028). When compared with those of CONT, circulating ghrelin concentrations of the SHAM treatment were greater at 15, 60, and 90 min after the introduction of visual, olfactory, and auditory cues at time 0 (Fig. 1; P < 0.05). Furthermore, the circulating ghrelin concentrations for SHAM were greater than those for FED at 30, 60, 90, and 120 min after feed was introduced (Fig. 1; P < 0.05). Additionally, circulating concentrations of ghrelin in the SHAM treatment were greater at time 15 than time at time −30. The FED treatment had greater ghrelin concentrations than CONT at −30 and −15 min (Fig. 1; P < 0.05). Additionally, the circulating concentrations of ghrelin in the FED treatment were greater at time −30 than times 30, 45, 60, 90, 105, and 120 and at time −15 than time 15 through 120. The AUC for plasma ghrelin concentrations before and after feed exposure demonstrated a group effect with a tendency toward significance such that SHAM ghrelin concentrations tended to be higher than FED ghrelin concentrations postfeeding (P = 0.0866; Fig. 2).

Figure 1.
Figure 1.

Average circulating concentrations of ghrelin in wethers that could eat offered 275 g of feed (FED; n = 5), wethers that were muzzled and could not eat offered 275 g of feed (SHAM; n = 4), and wethers that were not offered feed (CONT; n = 5). Arrows indicate the time FED and SHAM wethers were offered feed. *SHAM mean plasma ghrelin concentrations differed from CONT (P < 0.05). +SHAM mean plasma ghrelin concentrations differed from FED (P < 0.05). #CONT mean plasma ghrelin concentrations differed from FED (P < 0.05). Additionally, plasma concentrations of ghrelin in the SHAM treatment were greater at time 15 than at time −30 and in the FED treatment were greater at time −30 than at times 30, 45, 60, 90, 105, and 120 and at time −15 than at time 15 through 120.

 
Figure 2.
Figure 2.

The area under the curve (AUC) of ghrelin concentrations before and after the presentation of feed demonstrated a group effect with a tendency toward significance in which circulating concentrations of ghrelin of SHAM wethers were higher than those of FED wethers postfeeding (P = 0.0866). FED = wethers that could eat offered 275 g of feed; SHAM = wethers that were muzzled and could not eat offered 275 g of feed; CONT = wethers that were not offered feed.

 

DISCUSSION

Nutritional status of ruminants has been known to affect the secretion of ghrelin surrounding mealtimes. Although ghrelin has been observed to exhibit a pulsatile secretion pattern (Bagnasco et al., 2002), specific preprandial increases and postprandial decreases of plasma ghrelin have been observed in both cattle and sheep (Sugino et al., 2002a; Wertz-Lutz et al., 2006). Although not considered in the present study, circulating ghrelin concentrations of sheep are also affected by diet composition and flavor (Sugino et al., 2010; Sajedianfard et al., 2012; Villalba et al., 2011). Furthermore, the photoperiod impacts the severity of ghrelin’s ability to modify feed intake in sheep (Harrison et al., 2008). Additionally, direct action of ghrelin on the pituitary appears to differ depending on season and nutritional status (Kirsz et al., 2014). However, because the regulation of ghrelin secretion in ruminants is not fully understood, this study tested the impact of visual, olfactory, and auditory cues on plasma ghrelin concentrations in wethers. Our results demonstrated that ghrelin concentrations in the SHAM group were greater than in the FED and CONT groups following exposure to the cues, which included seeing and hearing the feed as it was placed in feed buckets and then smelling it while not being able to consume it. In addition to greater plasma ghrelin concentrations, the cues resulted in SHAM wethers becoming visibly agitated. Unlike the FED wethers, plasma ghrelin concentrations in the SHAM group were elevated after they were exposed to feed.

Previous research in rats has found that acute psychological stress results in elevated concentrations of circulating ghrelin (Kristenssson et al., 2006). Moreover, Verbeek et al. (2014) found that when ghrelin is administered exogenously, sheep tend to have elevated cortisol levels, thus suggesting a relationship between cortisol and ghrelin. Because the wethers participating in this study were not previously adapted or exposed to the muzzles yet SHAM wethers were fitted with muzzles before drawing the first blood sample at −30 min, the novelty of the muzzle itself may have induced a stress response in the SHAM and FED wethers. Moreover, the relative increase of FED circulating ghrelin concentrations before 0 min may be attributed to the stress inflicted by seeing muzzled SHAM wethers. Regardless, the observed differences in ghrelin concentrations were only significant (P < 0.05) between FED and CONT wethers at −30 and −15 min.

The observation that the SHAM group had elevated ghrelin concentrations after feed exposure was consistent with prior research indicating that ghrelin secretion has a neurological basis. Sugino et al. (2002a) demonstrated that plasma ghrelin concentrations in scheduled meal–fed sheep are subject to a preprandial rise and a postprandial decline. Alternatively, concentrations of circulating ghrelin in sheep fed ad libitum remain relatively constant and low throughout the sampling period (Sugino et al., 2002b). A later experiment by Simonian et al. (2005) found that human subjects receiving simulated meals also experienced an increase in circulating ghrelin concentrations before food exposure that was enabled via the stimulation of the vagal efferent nerve due to an anticipatory response. More recently, Schüssler et al. (2012) found that human circulating ghrelin concentrations increase after subjects are shown photographs of hedonic food. Before this study, however, such a response had not been demonstrated in sheep. Therefore, the rise of SHAM wether circulating ghrelin concentrations observed in the present study on exposure to visual, audio, and olfactory feeding cues suggests that ghrelin secretion by the autonomic nervous system is affected by the central nervous system.

The ability of plasma ghrelin to stimulate feed intake appears to be mediated by the central nervous system in the arcuate nucleus of the hypothalamus (Nakazato et al., 2001). One explanation has been that vagal nerve stimulation relays messages to the arcuate nucleus. Contained within the arcuate nucleus are agouti-related protein/neuropeptide Y neurons that are known to produce agouti-related protein and neuropeptide Y peptides (Hahn et al., 1998). Further research with mice by Chen et al. (2004)showed that disabling neuropeptide Y and agouti-related related protein reduces ghrelin-mediated feed intake. When administered via intracerebroventricular injections, neuropeptide Y and agouti-related protein increase feed intake in sheep (Wagner et al., 2004; Whitlock et al., 2005). Therefore, these neuropeptides may mediate ghrelin signaling to initiate feed consumption. The hormone leptin, which is produced by adipose tissue, suppresses feed intake in sheep via neuropeptide Y neurons (Bernard et al., 1999; Williams et al., 1999; Morrison et al., 2001). Studies also indicate that its capacity to decrease appetite is reduced in the presence of ghrelin, implying an antagonistic relationship between the 2 hormones (Shintani et al., 2001; Cummings and Foster, 2003). However, to ascertain if ovine ghrelin acts through this mechanism, further studies must be conducted using ruminant models.

Conclusion

In the present study, the authors worked to determine the effect of visual, olfactory, and auditory cues on peripheral ghrelin concentrations in scheduled meal–fed wethers outside of scheduled feeding times. Although denied satiation, SHAM wethers were exposed to the visual, olfactory, and auditory cues associated with feeding. Results indicated that plasma ghrelin concentrations in SHAM wethers were greater than those in CONT and FED wethers. This suggests that circulating ghrelin concentrations in scheduled meal–fed wethers are influenced by external cues. However, this study was designed to determine only the collective effect of visual, olfactory, and auditory cues. Further research will be required to discern the effect of each individual external cue on the peripheral ghrelin concentrations of scheduled meal–fed wethers.

 

References

Footnotes


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