Effect of short- and long-term feed restriction on ghrelin concentrations in turkeys

Effect of short- and long-term feed restriction on ghrelin concentrations in turkeys ABSTRACT One-day-old broad-breasted white turkeys (Meleagris gallopavo) were reared as recommended by industry standards. In Experiment 1, starting at 5 wk of age (WOA), birds were placed in individual cages with free access to feed and water. Blood samples were taken after 18 h of fasting (FASTING) and at 90 ± 5 min after feeding (1.5 h after feeding). In Experiment 2, birds were weighed, randomly assigned to 2 treatments, and placed in individual cages. In treatment 1 (n = 10), birds were fed ad libitum (FF), while birds in treatment 2 (n = 11) were placed on a restricted diet to allow for an average daily gain of 10.0 g per d from 4 to 11 WOA (RES). In Experiment 1, concentrations of ghrelin (P = 0.012) and glucose (P < 0.001) were increased 1.5 h after feeding compared with concentration during FASTING, whereas concentrations of adrenocorticotropic hormone (ACTH) (P < 0.001) and corticosterone (P = 0.002) were decreased 1.5 h after feeding. Concentration of insulin, free fatty acids, and ketone bodies followed a normal physiological response to fasting and feeding. Similarly, in Experiment 2, concentrations of ghrelin (P < 0.001) and glucose (P = 0.038) were increased in FF birds, whereas concentrations of corticosterone were decreased (P = 0.002) in FF birds. It could be concluded that in turkeys, preprandial (18 h of fasting) and long-term feed restriction is associated with decreased concentration of ghrelin—thus, the opposite effect of that reported in chickens and mammalian species. INTRODUCTION Ghrelin was discovered as the endogenous ligand for the “orphan” growth hormone secretagogue receptor in mammals (Kojima et al., 1999) and later in birds (Kaiya et al., 2002). The turkey preproghrelin gene consists of 5 exons and 4 introns, which is translated into a preproghrelin precursor protein (116 amino acids). The preproghrelin precursor, through several steps of cleavage, results in a final mature ghrelin peptide of 28 amino acids (Richards et al., 2006; Kojima and Kangawa, 2010). Thus, turkey ghrelin has 2 more amino acids (due to a proline extension at the C-terminal) than that of chicken and other avian species (Richards et al., 2006). In both mammals and birds, ghrelin is predominantly synthesized in the gastrointestinal tract and at lower amounts in the pancreas, hypothalamus, pituitary gland, and the lungs (Wada et al., 2003; Neglia et al., 2005; Chen et al., 2007). In mammals, systemic and intracerebroventricular (ICV) administration of ghrelin induces adiposity by stimulating an acute increase in feed intake, as well as a reduction in fat utilization (Tschop et al., 2000; Hayashida et al., 2001; Wren et al., 2001). However, in chickens and Japanese quails, ICV infusion of ghrelin inhibits feed intake (Furuse et al., 2001; Saito et al., 2002; Shousha et al., 2005). In turkeys, peripheral administration of ghrelin increases corticosterone and glucose concentrations (Aghdam Shahryar and Lotfi, 2017), whereas passive immunization against ghrelin increases feed intake (Vizcarra et al., 2012). The differential form of action of ghrelin in mammalian and avian species is thought to be associated with the activation of different neurons in the hypothalamus. In mammals, the effect of ghrelin on feed intake is linked with the activation of neurons expressing neuropeptides, such as neuropeptide Y (NPY) (Kojima and Kangawa, 2005). However, in birds, ghrelin is not associated with the activation of NPY. In chickens, the effect of ghrelin on feed intake is thought to be associated with the activation of neurons expressing corticotropin-releasing hormone (CRH), AMPK, γ-aminobutyric acid (GABA), nitric oxide (NO), urocortin, and β2 adrenergic receptors (Saito et al., 2005; Xu et al., 2011; Jonaidi et al., 2012; Kaiya et al., 2013; Zendehdel and Hassanpour, 2014). Preprandial rise in plasma ghrelin has been observed in humans (Cummings et al., 2001), cattle (Hayashida et al., 2001), rodents (Tschop et al., 2000), and pigs (Barretero-Hernandez et al., 2010). In contrast to mammals, conflicting and scant information is available on the effect of fasting and feeding on ghrelin concentrations in turkeys and other avian species. Previous studies have shown that in chickens and Japanese quail, concentrations of ghrelin increase during feed restriction (Kaiya et al., 2007; Xu et al., 2011). Concentration of ghrelin was higher during fasting compared with 6 h after feeding in six-day-old chickens (Kaiya et al., 2007). On the other hand, no differences in ghrelin concentrations were observed in three-week-old chickens that were exposed to fasting and re-feeding (Richards et al., 2006). The long- and short-term physiological response of ghrelin to feed restriction in other avian species such as turkeys has not been evaluated, to the best of our knowledge. Therefore, the objectives of this research were to evaluate the short- and long-term effects of fasting and re-feeding on ghrelin, glucose, corticosterone, and other metabolites in turkeys. MATERIALS AND METHODS Animal Rearing One-day-old broad-breasted white turkeys (Meleagris gallopavo) were reared using normal feeding and lighting management recommended by the industry. Briefly, from 1 to 14 d of age, birds were provided a standard corn-soybean-based starter diet followed by a grower/finisher diet until the end of the experiments (Purina, St. Louis, MO). At 3 wk of age (WOA), birds were transferred to individual cages (0.65 × 0.4 × 0.4 m) with continued free access to feed and water and a 23L:1D photoperiod. The care, treatment, and experimental protocols were approved by the Institutional Animal Care and Use Committee of Alabama A&M University. Experiment 1 At 5, 7, and 9 WOA, birds (n = 15) were weighed, and after 4 d of adaptation to the cages, feed was removed and animals were fasted for 18 h (FASTING). After fasting, a blood sample was obtained from each animal. Subsequently, all animals were fed, and a second blood sample was obtained 90 ± 5 min after feeding (1.5 h AFTER FEEDING). Feed intake was measured by recording the weight of feed offered minus any unconsumed feed remaining at 1.5 h after feeding. At the end of the experiment, birds were returned to their regular house in the poultry building. Experiment 2 At 2 WOA, birds were weighed, and after 7 d of adaptation to the cages, turkeys were randomly assigned to 2 treatments. In group treatment 1 (n = 10), birds were fed ad libitum (FF), while birds in group treatment 2 (n = 11) were placed on a restricted diet (RES). Initially, the restricted feeding regimen consisted of one-third of the quantity of feed per unit of body weight given to birds in the FF treatment. Subsequently, adjustments were made to allow for an average daily gain of 10.0 g per d from 4 to 12 WOA. Birds were fed daily at 0800 h, and daily feed intake was measured by recording the weight of feed offered each d minus any unconsumed feed remaining the next day. Blood samples were obtained once a wk (1100 h) beginning at 4 WOA until the end of the experiment (12 WOA). Body weight was recorded weekly. Blood Sampling Blood samples were collected via venipuncture of the brachial vein in 5-mL Vacutainer tubes containing EDTA (Fisher Scientific, Pittsburgh, PA). Immediately after blood collection, aprotinin (500 kIU/mL of blood) was added to the collection tube to inhibit the activity of proteinases. Tubes were gently rocked several times, placed on ice, and centrifuged within 1 hour. After centrifugation, plasma samples were labeled and stored at –80 °C in cryogenic tubes until concentrations of various hormones and metabolites were determined. Concentrations of corticosterone were evaluated using a RIA as previously described in our laboratory (Vizcarra et al., 2004). Concentrations of insulin, total ghrelin, adrenocorticotropic hormone (ACTH), and free fatty acids were evaluated using ELISA kits developed for chickens (Cusabio Biotech, College Park, MD). The kit used to evaluate total plasma ghrelin (CSB-E14230C) had an average recovery of mass of 94%, and when different volumes of sample were assayed, concentrations were parallel to the standard curve according to manufacturer's information. All samples were run in duplicates in one 96-well assay, thus only intra-assay coefficients of variation (CV) are reported (insulin sensitivity: 3.125 μIU/mL CV: 10%; total ghrelin sensitivity: 12.5 pg/mL; CV: 12% ACTH sensitivity: 0.125 pg/mL CV: 11%; free fatty acids sensitivity: 0.078 nmol/mL CV: 15%). Glucose (Cayman Chemical, Ann Arbor, MI) and ketone bodies (β-hydroxybutyrate; Cayman Chemical Co.) were evaluated using colorimetric procedures. Statistical Analysis An analysis of variance [PROC MIXED; (SAS, 2014)] was used to evaluate the effect of treatment on the concentrations of hormones and metabolites in Experiment 1. The statistical model included the effect of treatment (fasting or 1.5 h after feeding), wk of age (5, 7, or 9), and the treatment x wk interaction. No treatment x wk interaction was observed in any measurements; thus, data were pooled, and least square means are presented. The effect of wk of age on body weight and feed intake (1.5 h after feeding) was analyzed using a t test [PROC TTEST (SAS, 2014)]. In Experiment 2, the effects of treatment (FF vs. RES) on body weight gain (wk 1 to 12), concentrations of ghrelin (wk 4 to 12), glucose (wk 4 to 8), and corticosterone (wk 4 to 10) in weekly samples were analyzed using repeated measurements over time [PROC MIXED (SAS, 2014)]. At least 3 covariance structures were evaluated (compound symmetry, unstructured, and autoregressive). The autoregressive structure, with spatial power, provided the best model-fit criteria. If a significant treatment x wk interaction existed, the SLICE option in SAS was used to test for significant difference between treatments at each time. In both experiments, the cage was considered the experimental unit. RESULTS Experiment 1 Body weights at 5, 7, 9 WOA were different (1180.5 ± 96.6, 1994.3 ± 163.2, and 2898.9 ± 237.2 g, respectively, P < 0.001). Similarly, feed intake at 1.5 h after feeding was different (P < 0.02) between WOA (49.8 ± 5.3, 81.2 ± 8.3, and 94.0 ± 12.8 g for 5, 7, 9 WOA, respectively). There was no week or wk x treatment interaction on concentrations of glucose or insulin. However, there was a normal physiological response to feed intake as indicated by the significant increase in glucose and insulin concentrations 1.5 h after feeding (Figure 1). There was no wk or wk x treatment interaction on concentrations of free fatty acids or ketone bodies. However, free fatty acids and ketone bodies were increased (P < 0.005) during fasting (Figure 2). There was no wk or wk x treatment interaction on concentrations of ghrelin, ACTH, or corticosterone. Concentrations of these hormones significantly changed during fasting compared with 1.5 h after feeding (Table 1). Ghrelin concentrations were increased (P = 0.012) 1.5 h after feeding. Figure 1. View largeDownload slide Concentrations of glucose and insulin in turkeys during fasting and 1.5 h after feeding (Experiment 1). There were no wk or wk x treatment interactions on concentrations of glucose or insulin, and values represent the mean ± SE average over weeks. Different letters within a hormone or metabolite indicate a significant difference (n = 15 per treatment; P < 0.001). Figure 1. View largeDownload slide Concentrations of glucose and insulin in turkeys during fasting and 1.5 h after feeding (Experiment 1). There were no wk or wk x treatment interactions on concentrations of glucose or insulin, and values represent the mean ± SE average over weeks. Different letters within a hormone or metabolite indicate a significant difference (n = 15 per treatment; P < 0.001). Figure 2. View largeDownload slide Concentrations of free fatty acids and ketone bodies in turkeys during fasting and 1.5 h after feeding. There were no wk or wk x treatment interactions on concentrations of metabolites, and values represent the mean ± SE average over weeks. Different letters within a metabolite indicate a significant difference (n = 15 per treatment; P < 0.005). Figure 2. View largeDownload slide Concentrations of free fatty acids and ketone bodies in turkeys during fasting and 1.5 h after feeding. There were no wk or wk x treatment interactions on concentrations of metabolites, and values represent the mean ± SE average over weeks. Different letters within a metabolite indicate a significant difference (n = 15 per treatment; P < 0.005). Table 1. Concentrations of ghrelin, ACTH, and corticosterone in turkeys during fasting and 1.5 h after feeding. There were no wk or wk x treatment interactions on concentrations of these hormones. Values represent averages over weeks (n = 15 per treatment). Hormone Fasting 1.5 h after feeding P = SEM Ghrelin (pg/mL) 52.3 61.0 0.012 3.21 ACTH (pg/mL) 0.19 0.14 <0.001 0.01 Corticosterone (ng/mL) 0.39 0.17 0.002 0.07 Hormone Fasting 1.5 h after feeding P = SEM Ghrelin (pg/mL) 52.3 61.0 0.012 3.21 ACTH (pg/mL) 0.19 0.14 <0.001 0.01 Corticosterone (ng/mL) 0.39 0.17 0.002 0.07 View Large Table 1. Concentrations of ghrelin, ACTH, and corticosterone in turkeys during fasting and 1.5 h after feeding. There were no wk or wk x treatment interactions on concentrations of these hormones. Values represent averages over weeks (n = 15 per treatment). Hormone Fasting 1.5 h after feeding P = SEM Ghrelin (pg/mL) 52.3 61.0 0.012 3.21 ACTH (pg/mL) 0.19 0.14 <0.001 0.01 Corticosterone (ng/mL) 0.39 0.17 0.002 0.07 Hormone Fasting 1.5 h after feeding P = SEM Ghrelin (pg/mL) 52.3 61.0 0.012 3.21 ACTH (pg/mL) 0.19 0.14 <0.001 0.01 Corticosterone (ng/mL) 0.39 0.17 0.002 0.07 View Large Experiment 2 Feed management was successful in achieving target average daily gain in restricted birds. There was a treatment x time interaction on body weight gain (P < 0001; Figure 3). Birds that were in ad libitum feed consumption (FF) gained 620 ± 35 g/wk as opposed to 68 ± 5 g/wk in RES birds from 4 to 12 WOA. On average, feed intake in FF birds was 0.175 kg/d, whereas RES birds were offered 0.068 kg/d from wk 4 to 12. Figure 3. View largeDownload slide Body weight of full-fed (FF) and restricted (RES) birds from wk 1 to 12 (Experiment 2). There was a treatment x time interaction on body weight gain (P < 0001). Values represent the mean ± SE average over weeks. Figure 3. View largeDownload slide Body weight of full-fed (FF) and restricted (RES) birds from wk 1 to 12 (Experiment 2). There was a treatment x time interaction on body weight gain (P < 0001). Values represent the mean ± SE average over weeks. There was not a treatment x time interaction on concentrations of ghrelin (Figure 4). However, there was a treatment effect (P < 0.001). Concentration of ghrelin in FF birds (89.4 ± 2.6 pg/mL) was greater than that observed in RES animals (68.7 ± 2.7 pg/mL). Similarly, there was a treatment effect (P = 0.038) on glucose concentrations (Figure 5). Concentration of glucose in FF birds (271.9 ± 12.8 mg/dL) was greater than that observed in RES animals (228.7 ± 13.2 mg/dL). There was a treatment x time interaction on concentrations of corticosterone (P = 0.002). Concentrations of corticosterone (Figure 6) were greater in RES birds (31.6 ± 5.6 ng/mL) than in FF (6.8 ± 5.3 ng/mL). Figure 4. View largeDownload slide Concentrations of ghrelin in full-fed (FF) and restricted (RES) birds from wk 4 to 12 (Experiment 2). There was not a treatment x time interaction on concentrations of ghrelin. However, there was a treatment effect (P < 0.001). Values represent the mean ± SE average over weeks. Figure 4. View largeDownload slide Concentrations of ghrelin in full-fed (FF) and restricted (RES) birds from wk 4 to 12 (Experiment 2). There was not a treatment x time interaction on concentrations of ghrelin. However, there was a treatment effect (P < 0.001). Values represent the mean ± SE average over weeks. Figure 5. View largeDownload slide Concentrations of glucose in full-fed (FF) and restricted (RES) birds from wk 4 to 8 (Experiment 2). There was not a treatment x time interaction on concentrations of glucose. However, there was a treatment effect (P = 0.038). Values represent the mean ± SE average over weeks. Figure 5. View largeDownload slide Concentrations of glucose in full-fed (FF) and restricted (RES) birds from wk 4 to 8 (Experiment 2). There was not a treatment x time interaction on concentrations of glucose. However, there was a treatment effect (P = 0.038). Values represent the mean ± SE average over weeks. Figure 6. View largeDownload slide Concentrations of corticosterone in full-fed (FF) and restricted (RES) birds from wk 4 to 10 (Experiment 2). There was a treatment x time interaction on concentrations of corticosterone (P = 0.002). Values represent the mean ± SE average over weeks. Figure 6. View largeDownload slide Concentrations of corticosterone in full-fed (FF) and restricted (RES) birds from wk 4 to 10 (Experiment 2). There was a treatment x time interaction on concentrations of corticosterone (P = 0.002). Values represent the mean ± SE average over weeks. DISCUSSION Differences in body weight at 5, 7, and 9 WOA in Experiment 1 and body weight gain in FF turkeys (Experiment 2) reflect the normal growth and feed intake in turkeys raised using normal industry standards (Rivera-Torres et al., 2011). Gluconeogenesis and glycogenesis are the main mechanisms that maintain blood glucose concentrations in poultry, whereas glucagon and insulin maintain euglycemia (Scanes, 2009). Although earlier studies with chickens failed to find significant changes in glucose and insulin concentrations after fasting (Langslow et al., 1970; Belo et al., 1976), most modern-type poultry exhibit a glucose and insulin metabolism that is similar to that observed in mammalian species (de Beer et al., 2008; Christensen et al., 2013). For instance, when pigs were fasted for 18 h, concentrations of glucose and insulin were significantly different as compared with concentrations 1.5 hours after feeding (Barretero-Hernandez et al., 2010). Similarly, a 34% increase in glucose concentrations were observed in birds at 1.5 h after feeding compared to those during fasting (Experiment 1) and a 19% increase in glucose concentrations in FF birds compared to those that were feed restricted (Experiment 2). Insulin resistance is a characteristic observed in chickens that lack the insulin-responsive glucose transporter GLUT4 (Seki et al., 2003). In the present Experiment 1, concentrations of insulin were 14% higher 1.5 h after feeding compared with fasting. Significant changes in the insulin A-chain between avian and mammalian species minimizes the ability of mammalian antibodies to cross-react with chicken insulin (Dupont et al., 2015). Thus, the use of specific chicken insulin antibodies used in the present experiment overcomes the potential cross-reactivity encounter in earlier works when mammalian antibodies were used. Fasting shifts the metabolic balance from lipogenesis to lipolysis (Scanes, 2015). Free fatty acids are released during fasting after the adipose triglyceride lipase enzyme, present in avian species, hydrolyzes the first ester bond in triglycerides (Serr et al., 2009). Subsequently, oxidative decarboxylation of fatty acids (β-oxidation) releases acetyl CoA, which serves as the substrate to produce ketone bodies. In the present Experiment 1, fasted birds had 54% more free fatty acids and 111% more ketone bodies compared with concentrations 1.5 h after feeding. Similarly, free fatty acids were significantly increased when turkey were fasted for up to 44 h (Bacon, 1986; Anthony et al., 1990). Thus, during fasting, lipid mobilization from subcutaneous fat tends to increase to provide non-gluconeogenic substrates in turkeys (de Beer et al., 2008; Serr et al., 2009). Taken together, concentrations of glucose, insulin, free fatty acids, and ketone bodies reflected the normal physiological response during fasting and re-feeding in turkeys used in Experiment 1. To the best of our knowledge, this is the first report that quantifies ghrelin concentrations in fasting and re-feeding turkeys (Experiment 1) and between FF and RES birds (Experiment 2). Data clearly indicate that postprandial and long-term feed restriction concentrations of ghrelin are the opposite of that reported in mammalian species, including pigs, as reported in our laboratory (Barretero-Hernandez et al., 2010). These results are in contrast with those previously reported in short-term experiments with chickens (Richards et al., 2006; Kaiya et al., 2007). The discrepancy between results in the present experiment and those reported elsewhere may be explained in part by the type of animal used (turkeys and chickens), and the use of assays developed for mammalian (Richards et al., 2006; Kaiya et al., 2007) and avian species reported here and elsewhere (Najafi et al., 2015; Yu et al., 2016; Hohne et al., 2017). Turkeys and chickens share a similar sequence of amino acids; the mature ghrelin peptide in chickens consists of 26 amino acids as opposed to 28 amino acids in turkeys. There is a high degree of similarity in the amino acid sequence of the preproghrlein precursors (93% homology) in chickens and turkeys (Richards et al., 2006). However, the turkey ghrelin precursor peptide possesses a Pro-Arg at the carboxyl-terminal processing site of the mature ghrelin peptide similar to its mammalian homologs. All other known avian species possess an Arg-Arg at the carboxyl-terminal (Kojima and Kangawa, 2005). Turkey and chicken ghrelins exhibit only 54% sequence identity with the human and rat orthologues (Kaiya et al., 2002; Richards and McMurtry, 2010); thus, the development of assays with antibodies that are specific for different species is essential to evaluate pre- and postprandial concentrations of ghrelin. Moreover, turkey and chicken ghrelins are peptides with an n-octanoylated modification (Richards et al., 2006; Kaiya et al., 2009). Octanoylation is a post-translational process catalyzed by ghrelin O-acyl transferase (GOAT), an enzyme that attaches n-octanoic acid to the amino residue Ser-3 (Gutierrez et al., 2008; Yang et al., 2008). Therefore, 2 forms of ghrelins are present in blood (acyl- and desacyl-ghrelin). In the present experiment, total ghrelin was measured. Robust avian assays that can distinguish between these 2 forms will certainly aid in the understanding of ghrelin in poultry. Nevertheless, the increased postprandial concentrations of ghrelin reported here confirm previous evidence that suggests that ghrelin is an anorixegenic hormone in avian species (Furuse et al., 2001; Saito et al., 2002; Xu et al., 2011). The physiological response to fasting on corticosterone concentrations reported here (Experiments 1 and 2) are in agreement with previous work performed in chickens (Richards et al., 2006; Kaiya et al., 2007; de Beer et al., 2008). We speculate that if CRH is mediating the release of ghrelin as previously reported in chicks (Saito et al., 2005), then the potential effect of stress associated with fasting overrode the effect of CRH (as measured by ACTH and corticosterone concentrations; see Table 1 and Figure 6) on ghrelin secretion. Although corticosterone concentrations were increased after ICV injections of ghrelin (Saito et al., 2005), other lines of research suggest that CRH may not be the main mediator of the orexigenic effect of ghrelin. 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Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Effect of short- and long-term feed restriction on ghrelin concentrations in turkeys

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© 2018 Poultry Science Association Inc.
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0032-5791
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Abstract

ABSTRACT One-day-old broad-breasted white turkeys (Meleagris gallopavo) were reared as recommended by industry standards. In Experiment 1, starting at 5 wk of age (WOA), birds were placed in individual cages with free access to feed and water. Blood samples were taken after 18 h of fasting (FASTING) and at 90 ± 5 min after feeding (1.5 h after feeding). In Experiment 2, birds were weighed, randomly assigned to 2 treatments, and placed in individual cages. In treatment 1 (n = 10), birds were fed ad libitum (FF), while birds in treatment 2 (n = 11) were placed on a restricted diet to allow for an average daily gain of 10.0 g per d from 4 to 11 WOA (RES). In Experiment 1, concentrations of ghrelin (P = 0.012) and glucose (P < 0.001) were increased 1.5 h after feeding compared with concentration during FASTING, whereas concentrations of adrenocorticotropic hormone (ACTH) (P < 0.001) and corticosterone (P = 0.002) were decreased 1.5 h after feeding. Concentration of insulin, free fatty acids, and ketone bodies followed a normal physiological response to fasting and feeding. Similarly, in Experiment 2, concentrations of ghrelin (P < 0.001) and glucose (P = 0.038) were increased in FF birds, whereas concentrations of corticosterone were decreased (P = 0.002) in FF birds. It could be concluded that in turkeys, preprandial (18 h of fasting) and long-term feed restriction is associated with decreased concentration of ghrelin—thus, the opposite effect of that reported in chickens and mammalian species. INTRODUCTION Ghrelin was discovered as the endogenous ligand for the “orphan” growth hormone secretagogue receptor in mammals (Kojima et al., 1999) and later in birds (Kaiya et al., 2002). The turkey preproghrelin gene consists of 5 exons and 4 introns, which is translated into a preproghrelin precursor protein (116 amino acids). The preproghrelin precursor, through several steps of cleavage, results in a final mature ghrelin peptide of 28 amino acids (Richards et al., 2006; Kojima and Kangawa, 2010). Thus, turkey ghrelin has 2 more amino acids (due to a proline extension at the C-terminal) than that of chicken and other avian species (Richards et al., 2006). In both mammals and birds, ghrelin is predominantly synthesized in the gastrointestinal tract and at lower amounts in the pancreas, hypothalamus, pituitary gland, and the lungs (Wada et al., 2003; Neglia et al., 2005; Chen et al., 2007). In mammals, systemic and intracerebroventricular (ICV) administration of ghrelin induces adiposity by stimulating an acute increase in feed intake, as well as a reduction in fat utilization (Tschop et al., 2000; Hayashida et al., 2001; Wren et al., 2001). However, in chickens and Japanese quails, ICV infusion of ghrelin inhibits feed intake (Furuse et al., 2001; Saito et al., 2002; Shousha et al., 2005). In turkeys, peripheral administration of ghrelin increases corticosterone and glucose concentrations (Aghdam Shahryar and Lotfi, 2017), whereas passive immunization against ghrelin increases feed intake (Vizcarra et al., 2012). The differential form of action of ghrelin in mammalian and avian species is thought to be associated with the activation of different neurons in the hypothalamus. In mammals, the effect of ghrelin on feed intake is linked with the activation of neurons expressing neuropeptides, such as neuropeptide Y (NPY) (Kojima and Kangawa, 2005). However, in birds, ghrelin is not associated with the activation of NPY. In chickens, the effect of ghrelin on feed intake is thought to be associated with the activation of neurons expressing corticotropin-releasing hormone (CRH), AMPK, γ-aminobutyric acid (GABA), nitric oxide (NO), urocortin, and β2 adrenergic receptors (Saito et al., 2005; Xu et al., 2011; Jonaidi et al., 2012; Kaiya et al., 2013; Zendehdel and Hassanpour, 2014). Preprandial rise in plasma ghrelin has been observed in humans (Cummings et al., 2001), cattle (Hayashida et al., 2001), rodents (Tschop et al., 2000), and pigs (Barretero-Hernandez et al., 2010). In contrast to mammals, conflicting and scant information is available on the effect of fasting and feeding on ghrelin concentrations in turkeys and other avian species. Previous studies have shown that in chickens and Japanese quail, concentrations of ghrelin increase during feed restriction (Kaiya et al., 2007; Xu et al., 2011). Concentration of ghrelin was higher during fasting compared with 6 h after feeding in six-day-old chickens (Kaiya et al., 2007). On the other hand, no differences in ghrelin concentrations were observed in three-week-old chickens that were exposed to fasting and re-feeding (Richards et al., 2006). The long- and short-term physiological response of ghrelin to feed restriction in other avian species such as turkeys has not been evaluated, to the best of our knowledge. Therefore, the objectives of this research were to evaluate the short- and long-term effects of fasting and re-feeding on ghrelin, glucose, corticosterone, and other metabolites in turkeys. MATERIALS AND METHODS Animal Rearing One-day-old broad-breasted white turkeys (Meleagris gallopavo) were reared using normal feeding and lighting management recommended by the industry. Briefly, from 1 to 14 d of age, birds were provided a standard corn-soybean-based starter diet followed by a grower/finisher diet until the end of the experiments (Purina, St. Louis, MO). At 3 wk of age (WOA), birds were transferred to individual cages (0.65 × 0.4 × 0.4 m) with continued free access to feed and water and a 23L:1D photoperiod. The care, treatment, and experimental protocols were approved by the Institutional Animal Care and Use Committee of Alabama A&M University. Experiment 1 At 5, 7, and 9 WOA, birds (n = 15) were weighed, and after 4 d of adaptation to the cages, feed was removed and animals were fasted for 18 h (FASTING). After fasting, a blood sample was obtained from each animal. Subsequently, all animals were fed, and a second blood sample was obtained 90 ± 5 min after feeding (1.5 h AFTER FEEDING). Feed intake was measured by recording the weight of feed offered minus any unconsumed feed remaining at 1.5 h after feeding. At the end of the experiment, birds were returned to their regular house in the poultry building. Experiment 2 At 2 WOA, birds were weighed, and after 7 d of adaptation to the cages, turkeys were randomly assigned to 2 treatments. In group treatment 1 (n = 10), birds were fed ad libitum (FF), while birds in group treatment 2 (n = 11) were placed on a restricted diet (RES). Initially, the restricted feeding regimen consisted of one-third of the quantity of feed per unit of body weight given to birds in the FF treatment. Subsequently, adjustments were made to allow for an average daily gain of 10.0 g per d from 4 to 12 WOA. Birds were fed daily at 0800 h, and daily feed intake was measured by recording the weight of feed offered each d minus any unconsumed feed remaining the next day. Blood samples were obtained once a wk (1100 h) beginning at 4 WOA until the end of the experiment (12 WOA). Body weight was recorded weekly. Blood Sampling Blood samples were collected via venipuncture of the brachial vein in 5-mL Vacutainer tubes containing EDTA (Fisher Scientific, Pittsburgh, PA). Immediately after blood collection, aprotinin (500 kIU/mL of blood) was added to the collection tube to inhibit the activity of proteinases. Tubes were gently rocked several times, placed on ice, and centrifuged within 1 hour. After centrifugation, plasma samples were labeled and stored at –80 °C in cryogenic tubes until concentrations of various hormones and metabolites were determined. Concentrations of corticosterone were evaluated using a RIA as previously described in our laboratory (Vizcarra et al., 2004). Concentrations of insulin, total ghrelin, adrenocorticotropic hormone (ACTH), and free fatty acids were evaluated using ELISA kits developed for chickens (Cusabio Biotech, College Park, MD). The kit used to evaluate total plasma ghrelin (CSB-E14230C) had an average recovery of mass of 94%, and when different volumes of sample were assayed, concentrations were parallel to the standard curve according to manufacturer's information. All samples were run in duplicates in one 96-well assay, thus only intra-assay coefficients of variation (CV) are reported (insulin sensitivity: 3.125 μIU/mL CV: 10%; total ghrelin sensitivity: 12.5 pg/mL; CV: 12% ACTH sensitivity: 0.125 pg/mL CV: 11%; free fatty acids sensitivity: 0.078 nmol/mL CV: 15%). Glucose (Cayman Chemical, Ann Arbor, MI) and ketone bodies (β-hydroxybutyrate; Cayman Chemical Co.) were evaluated using colorimetric procedures. Statistical Analysis An analysis of variance [PROC MIXED; (SAS, 2014)] was used to evaluate the effect of treatment on the concentrations of hormones and metabolites in Experiment 1. The statistical model included the effect of treatment (fasting or 1.5 h after feeding), wk of age (5, 7, or 9), and the treatment x wk interaction. No treatment x wk interaction was observed in any measurements; thus, data were pooled, and least square means are presented. The effect of wk of age on body weight and feed intake (1.5 h after feeding) was analyzed using a t test [PROC TTEST (SAS, 2014)]. In Experiment 2, the effects of treatment (FF vs. RES) on body weight gain (wk 1 to 12), concentrations of ghrelin (wk 4 to 12), glucose (wk 4 to 8), and corticosterone (wk 4 to 10) in weekly samples were analyzed using repeated measurements over time [PROC MIXED (SAS, 2014)]. At least 3 covariance structures were evaluated (compound symmetry, unstructured, and autoregressive). The autoregressive structure, with spatial power, provided the best model-fit criteria. If a significant treatment x wk interaction existed, the SLICE option in SAS was used to test for significant difference between treatments at each time. In both experiments, the cage was considered the experimental unit. RESULTS Experiment 1 Body weights at 5, 7, 9 WOA were different (1180.5 ± 96.6, 1994.3 ± 163.2, and 2898.9 ± 237.2 g, respectively, P < 0.001). Similarly, feed intake at 1.5 h after feeding was different (P < 0.02) between WOA (49.8 ± 5.3, 81.2 ± 8.3, and 94.0 ± 12.8 g for 5, 7, 9 WOA, respectively). There was no week or wk x treatment interaction on concentrations of glucose or insulin. However, there was a normal physiological response to feed intake as indicated by the significant increase in glucose and insulin concentrations 1.5 h after feeding (Figure 1). There was no wk or wk x treatment interaction on concentrations of free fatty acids or ketone bodies. However, free fatty acids and ketone bodies were increased (P < 0.005) during fasting (Figure 2). There was no wk or wk x treatment interaction on concentrations of ghrelin, ACTH, or corticosterone. Concentrations of these hormones significantly changed during fasting compared with 1.5 h after feeding (Table 1). Ghrelin concentrations were increased (P = 0.012) 1.5 h after feeding. Figure 1. View largeDownload slide Concentrations of glucose and insulin in turkeys during fasting and 1.5 h after feeding (Experiment 1). There were no wk or wk x treatment interactions on concentrations of glucose or insulin, and values represent the mean ± SE average over weeks. Different letters within a hormone or metabolite indicate a significant difference (n = 15 per treatment; P < 0.001). Figure 1. View largeDownload slide Concentrations of glucose and insulin in turkeys during fasting and 1.5 h after feeding (Experiment 1). There were no wk or wk x treatment interactions on concentrations of glucose or insulin, and values represent the mean ± SE average over weeks. Different letters within a hormone or metabolite indicate a significant difference (n = 15 per treatment; P < 0.001). Figure 2. View largeDownload slide Concentrations of free fatty acids and ketone bodies in turkeys during fasting and 1.5 h after feeding. There were no wk or wk x treatment interactions on concentrations of metabolites, and values represent the mean ± SE average over weeks. Different letters within a metabolite indicate a significant difference (n = 15 per treatment; P < 0.005). Figure 2. View largeDownload slide Concentrations of free fatty acids and ketone bodies in turkeys during fasting and 1.5 h after feeding. There were no wk or wk x treatment interactions on concentrations of metabolites, and values represent the mean ± SE average over weeks. Different letters within a metabolite indicate a significant difference (n = 15 per treatment; P < 0.005). Table 1. Concentrations of ghrelin, ACTH, and corticosterone in turkeys during fasting and 1.5 h after feeding. There were no wk or wk x treatment interactions on concentrations of these hormones. Values represent averages over weeks (n = 15 per treatment). Hormone Fasting 1.5 h after feeding P = SEM Ghrelin (pg/mL) 52.3 61.0 0.012 3.21 ACTH (pg/mL) 0.19 0.14 <0.001 0.01 Corticosterone (ng/mL) 0.39 0.17 0.002 0.07 Hormone Fasting 1.5 h after feeding P = SEM Ghrelin (pg/mL) 52.3 61.0 0.012 3.21 ACTH (pg/mL) 0.19 0.14 <0.001 0.01 Corticosterone (ng/mL) 0.39 0.17 0.002 0.07 View Large Table 1. Concentrations of ghrelin, ACTH, and corticosterone in turkeys during fasting and 1.5 h after feeding. There were no wk or wk x treatment interactions on concentrations of these hormones. Values represent averages over weeks (n = 15 per treatment). Hormone Fasting 1.5 h after feeding P = SEM Ghrelin (pg/mL) 52.3 61.0 0.012 3.21 ACTH (pg/mL) 0.19 0.14 <0.001 0.01 Corticosterone (ng/mL) 0.39 0.17 0.002 0.07 Hormone Fasting 1.5 h after feeding P = SEM Ghrelin (pg/mL) 52.3 61.0 0.012 3.21 ACTH (pg/mL) 0.19 0.14 <0.001 0.01 Corticosterone (ng/mL) 0.39 0.17 0.002 0.07 View Large Experiment 2 Feed management was successful in achieving target average daily gain in restricted birds. There was a treatment x time interaction on body weight gain (P < 0001; Figure 3). Birds that were in ad libitum feed consumption (FF) gained 620 ± 35 g/wk as opposed to 68 ± 5 g/wk in RES birds from 4 to 12 WOA. On average, feed intake in FF birds was 0.175 kg/d, whereas RES birds were offered 0.068 kg/d from wk 4 to 12. Figure 3. View largeDownload slide Body weight of full-fed (FF) and restricted (RES) birds from wk 1 to 12 (Experiment 2). There was a treatment x time interaction on body weight gain (P < 0001). Values represent the mean ± SE average over weeks. Figure 3. View largeDownload slide Body weight of full-fed (FF) and restricted (RES) birds from wk 1 to 12 (Experiment 2). There was a treatment x time interaction on body weight gain (P < 0001). Values represent the mean ± SE average over weeks. There was not a treatment x time interaction on concentrations of ghrelin (Figure 4). However, there was a treatment effect (P < 0.001). Concentration of ghrelin in FF birds (89.4 ± 2.6 pg/mL) was greater than that observed in RES animals (68.7 ± 2.7 pg/mL). Similarly, there was a treatment effect (P = 0.038) on glucose concentrations (Figure 5). Concentration of glucose in FF birds (271.9 ± 12.8 mg/dL) was greater than that observed in RES animals (228.7 ± 13.2 mg/dL). There was a treatment x time interaction on concentrations of corticosterone (P = 0.002). Concentrations of corticosterone (Figure 6) were greater in RES birds (31.6 ± 5.6 ng/mL) than in FF (6.8 ± 5.3 ng/mL). Figure 4. View largeDownload slide Concentrations of ghrelin in full-fed (FF) and restricted (RES) birds from wk 4 to 12 (Experiment 2). There was not a treatment x time interaction on concentrations of ghrelin. However, there was a treatment effect (P < 0.001). Values represent the mean ± SE average over weeks. Figure 4. View largeDownload slide Concentrations of ghrelin in full-fed (FF) and restricted (RES) birds from wk 4 to 12 (Experiment 2). There was not a treatment x time interaction on concentrations of ghrelin. However, there was a treatment effect (P < 0.001). Values represent the mean ± SE average over weeks. Figure 5. View largeDownload slide Concentrations of glucose in full-fed (FF) and restricted (RES) birds from wk 4 to 8 (Experiment 2). There was not a treatment x time interaction on concentrations of glucose. However, there was a treatment effect (P = 0.038). Values represent the mean ± SE average over weeks. Figure 5. View largeDownload slide Concentrations of glucose in full-fed (FF) and restricted (RES) birds from wk 4 to 8 (Experiment 2). There was not a treatment x time interaction on concentrations of glucose. However, there was a treatment effect (P = 0.038). Values represent the mean ± SE average over weeks. Figure 6. View largeDownload slide Concentrations of corticosterone in full-fed (FF) and restricted (RES) birds from wk 4 to 10 (Experiment 2). There was a treatment x time interaction on concentrations of corticosterone (P = 0.002). Values represent the mean ± SE average over weeks. Figure 6. View largeDownload slide Concentrations of corticosterone in full-fed (FF) and restricted (RES) birds from wk 4 to 10 (Experiment 2). There was a treatment x time interaction on concentrations of corticosterone (P = 0.002). Values represent the mean ± SE average over weeks. DISCUSSION Differences in body weight at 5, 7, and 9 WOA in Experiment 1 and body weight gain in FF turkeys (Experiment 2) reflect the normal growth and feed intake in turkeys raised using normal industry standards (Rivera-Torres et al., 2011). Gluconeogenesis and glycogenesis are the main mechanisms that maintain blood glucose concentrations in poultry, whereas glucagon and insulin maintain euglycemia (Scanes, 2009). Although earlier studies with chickens failed to find significant changes in glucose and insulin concentrations after fasting (Langslow et al., 1970; Belo et al., 1976), most modern-type poultry exhibit a glucose and insulin metabolism that is similar to that observed in mammalian species (de Beer et al., 2008; Christensen et al., 2013). For instance, when pigs were fasted for 18 h, concentrations of glucose and insulin were significantly different as compared with concentrations 1.5 hours after feeding (Barretero-Hernandez et al., 2010). Similarly, a 34% increase in glucose concentrations were observed in birds at 1.5 h after feeding compared to those during fasting (Experiment 1) and a 19% increase in glucose concentrations in FF birds compared to those that were feed restricted (Experiment 2). Insulin resistance is a characteristic observed in chickens that lack the insulin-responsive glucose transporter GLUT4 (Seki et al., 2003). In the present Experiment 1, concentrations of insulin were 14% higher 1.5 h after feeding compared with fasting. Significant changes in the insulin A-chain between avian and mammalian species minimizes the ability of mammalian antibodies to cross-react with chicken insulin (Dupont et al., 2015). Thus, the use of specific chicken insulin antibodies used in the present experiment overcomes the potential cross-reactivity encounter in earlier works when mammalian antibodies were used. Fasting shifts the metabolic balance from lipogenesis to lipolysis (Scanes, 2015). Free fatty acids are released during fasting after the adipose triglyceride lipase enzyme, present in avian species, hydrolyzes the first ester bond in triglycerides (Serr et al., 2009). Subsequently, oxidative decarboxylation of fatty acids (β-oxidation) releases acetyl CoA, which serves as the substrate to produce ketone bodies. In the present Experiment 1, fasted birds had 54% more free fatty acids and 111% more ketone bodies compared with concentrations 1.5 h after feeding. Similarly, free fatty acids were significantly increased when turkey were fasted for up to 44 h (Bacon, 1986; Anthony et al., 1990). Thus, during fasting, lipid mobilization from subcutaneous fat tends to increase to provide non-gluconeogenic substrates in turkeys (de Beer et al., 2008; Serr et al., 2009). Taken together, concentrations of glucose, insulin, free fatty acids, and ketone bodies reflected the normal physiological response during fasting and re-feeding in turkeys used in Experiment 1. To the best of our knowledge, this is the first report that quantifies ghrelin concentrations in fasting and re-feeding turkeys (Experiment 1) and between FF and RES birds (Experiment 2). Data clearly indicate that postprandial and long-term feed restriction concentrations of ghrelin are the opposite of that reported in mammalian species, including pigs, as reported in our laboratory (Barretero-Hernandez et al., 2010). These results are in contrast with those previously reported in short-term experiments with chickens (Richards et al., 2006; Kaiya et al., 2007). The discrepancy between results in the present experiment and those reported elsewhere may be explained in part by the type of animal used (turkeys and chickens), and the use of assays developed for mammalian (Richards et al., 2006; Kaiya et al., 2007) and avian species reported here and elsewhere (Najafi et al., 2015; Yu et al., 2016; Hohne et al., 2017). Turkeys and chickens share a similar sequence of amino acids; the mature ghrelin peptide in chickens consists of 26 amino acids as opposed to 28 amino acids in turkeys. There is a high degree of similarity in the amino acid sequence of the preproghrlein precursors (93% homology) in chickens and turkeys (Richards et al., 2006). However, the turkey ghrelin precursor peptide possesses a Pro-Arg at the carboxyl-terminal processing site of the mature ghrelin peptide similar to its mammalian homologs. All other known avian species possess an Arg-Arg at the carboxyl-terminal (Kojima and Kangawa, 2005). Turkey and chicken ghrelins exhibit only 54% sequence identity with the human and rat orthologues (Kaiya et al., 2002; Richards and McMurtry, 2010); thus, the development of assays with antibodies that are specific for different species is essential to evaluate pre- and postprandial concentrations of ghrelin. Moreover, turkey and chicken ghrelins are peptides with an n-octanoylated modification (Richards et al., 2006; Kaiya et al., 2009). Octanoylation is a post-translational process catalyzed by ghrelin O-acyl transferase (GOAT), an enzyme that attaches n-octanoic acid to the amino residue Ser-3 (Gutierrez et al., 2008; Yang et al., 2008). Therefore, 2 forms of ghrelins are present in blood (acyl- and desacyl-ghrelin). In the present experiment, total ghrelin was measured. Robust avian assays that can distinguish between these 2 forms will certainly aid in the understanding of ghrelin in poultry. Nevertheless, the increased postprandial concentrations of ghrelin reported here confirm previous evidence that suggests that ghrelin is an anorixegenic hormone in avian species (Furuse et al., 2001; Saito et al., 2002; Xu et al., 2011). The physiological response to fasting on corticosterone concentrations reported here (Experiments 1 and 2) are in agreement with previous work performed in chickens (Richards et al., 2006; Kaiya et al., 2007; de Beer et al., 2008). We speculate that if CRH is mediating the release of ghrelin as previously reported in chicks (Saito et al., 2005), then the potential effect of stress associated with fasting overrode the effect of CRH (as measured by ACTH and corticosterone concentrations; see Table 1 and Figure 6) on ghrelin secretion. Although corticosterone concentrations were increased after ICV injections of ghrelin (Saito et al., 2005), other lines of research suggest that CRH may not be the main mediator of the orexigenic effect of ghrelin. In fact, recent reports suggested that AMPK, GABA, NO, urocortin, and β2 adrenergic receptors also mediate the decrease in feed intake associated with ICV injections of ghrelin (Xu et al., 2011; Jonaidi et al., 2012; Kaiya et al., 2013; Zendehdel and Hassanpour, 2014). We concluded that in turkeys, preprandial (18 h of fasting) and long-term feed restriction is associated with decreased concentration of ghrelin—thus, the opposite effect of that reported in chickens and mammals. Nonetheless, concentrations of insulin, glucose, free fatty acids, ketone bodies, ACTH, and corticosterone follow the normal physiological response to fasting and feeding observed in avian and mammalian species. Footnotes 1 This project was supported by Agriculture and Food Research Initiative Competitive Grant # 2016-67016-24945 from the USDA National Institute of Food and Agriculture. REFERENCES Aghdam Shahryar H. , Lotfi A. . 2017 . 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Poultry ScienceOxford University Press

Published: Feb 28, 2018

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