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1. The present study compared the effects of fasting on circulating concentrations of glucose, insulin and glucagon in male and female modern meat-type chickens (Ross 708) at three ages (19 d, 33 d and 47 d). 2. Plasma concentrations of glucose were reduced by fasting with reductions of 24.9% (19-d-old), 22.6% (33-d-old) and 17.9% (47-d-old) in broiler chickens fasted for 12 h. 3. Plasma concentrations of insulin decreased with fasting. For instance, circulating concentrations of insulin declined after 6 h of fasting by 45.7%, 54.7% and 50.0%, respectively, in 19-d-old, 33-d-old and 47-d-old broiler chickens. 4. Plasma concentrations of glucagon were increased by fasting. Plasma concentrations of glucagon were elevated by 3.79% (19-d-old), 3.51% (33-d-old) and 3.79% (47-d-old) with 6 h of fasting and remained elevated with 12 h, 18 h and 24 h of fasting.

The aim of this study is to elucidate whether insulin acts differentially within the central nervous system (CNS) of two types of commercial chicks to control ingestive behavior. Male layer and broiler chicks (4-day-old) were intracerebroventricularly (ICV) injected with saline or insulin under satiated and starved conditions. Feed intake was measured at 30, 60 and 120 min after treatment. Secondly, blood and hypothalamus were collected from both chick types under ad libitum feeding and fasting for 24 h. Plasma insulin concentration was measured by time-resolved fluoro-immunoassay. Hypothalamic insulin receptor mRNA expression levels were measured by quantitative RT-PCR. The ICV injection of insulin significantly inhibited feed consumption in layer chicks when compared with saline (P<0.05), but not broiler chicks (P>0.1). Plasma insulin concentration of both chick types significantly decreased following 24 h of fasting, while insulin concentrations in the broiler chicks were significantly higher compared to the layers fed under ad libitum conditions. Hypothalamic insulin receptor mRNA expression levels were significantly lower (P<0.05) in broiler chicks than in layer ones under ad libitum feeding. Feed deprivation significantly decreased insulin receptor mRNA levels in layer chicks (P<0.01), but not in broiler chicks (P>0.1). Moreover, plasma insulin concentrations correlated negatively with hypothalamic insulin receptor protein expression in the two types of chicks fed ad libitum (P<0.05). These results suggest that insulin resistance exists in the CNS of broiler chicks, possibly due to persistent hyperinsulinemia, which results in a down-regulation of CNS insulin receptor expression compared to that in layer chicks., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The embryo to neonate transition is a critical period of development that has significant impact on broiler production. During this time important genetic programs governing metabolism and growth are established. The goal of this work was to study the effects of early post-hatch (PH) development and the time of initiation of feeding on activation of the genetic program regulating hepatic lipogenesis. A comparison of liver total RNA samples at hatch and 7 days PH was performed using oligonucleotide-based (Affymetrix GeneChip®) chicken genome microarrays. During the first week PH there was significant up-regulation of key lipogenic genes including: ATP citrate lyase (ACL), malic enzyme (ME), fatty acid synthase (FAS), acetyl-CoA carboxylase alpha (ACCα), stearoyl-CoA desaturase-1 (SCD-1), sterol regulatory element binding protein-2 (SREBP-2) and thyroid hormone responsive spot 14α (Spot 14α) among others. These findings were confirmed using gene-specific RT-PCR assays. In a follow-up study, we investigated the effects of withholding feed for the first 48 h PH (delayed feeding, DF) on lipogenic gene expression through 8 days PH. Body weight gain was significantly depressed by DF. Plasma levels of the major metabolic hormones that regulate lipogenic gene expression (insulin, glucagon and T(3)) changed significantly during PH development, but were largely unaffected by DF. Plasma glucose was significantly lower in the DF group at 24h PH but recovered thereafter. In general, DF inhibited the up-regulation of lipogenic genes until feeding was initiated. Delayed up-regulation was also observed for the lipogenic transcription factor genes, SREBP-1, SREBP-2 and peroxisome proliferator-activated receptor gamma (PPARγ), but not for carbohydrate response element binding protein (ChREB) or liver X receptor (LXR). Our results offer additional insight into the transcriptional programming of hepatic lipogenesis in response to the transition from high fat (yolk) to high carbohydrate (feed) nutrition that occurs during early PH development., (Published by Elsevier Inc.)

Research addressing digestible Lys requirement data of modern broilers from 4 to 6 wk of age is limited. Male broilers (1,632 Ross×Ross TP16 and 3,000 Cobb×Cobb 700) were used in separate experiments to determine the digestible Lys requirements from 28 to 42 d. In each experiment, 2 diets (dilution and summit) consisting of corn, soybean meal, animal protein meal, and peanut meal were formulated to be adequate in all other amino acids. The dilution and summit diets were blended to create 9 titration diets. A control diet formulated to contain corn, soybean meal, and animal protein meal as the primary ingredients was used for comparison with the titration diets. Body weight gain, feed intake, digestible Lys intake, digestible Lys intake:BW gain, feed conversion, mortality, carcass yields, and physiological measurements were assessed during experimentation. Digestible Lys requirements were estimated using a quadratic broken-line model. In experiment 1, the digestible Lys requirement for male Ross×Ross TP16 broilers was determined at 0.988, 1.053, 0.939, and 0.962%, respectively, for BW gain, feed conversion, carcass weight, and total breast meat weight. In experiment 2, the digestible Lys requirement for male Cobb×Cobb 700 broilers ranged from 0.965, 1.012, 1.029, 0.987, and 0.981%, respectively, for 28- to 42-d BW gain, feed conversion, carcass weight, total breast meat weight, and total breast meat yield. Digestible Lys requirements for male Ross×Ross TP16 and Cobb×Cobb 700 broilers were estimated at 1.001 and 0.995%, respectively, based upon averages of live performance and meat yield responses. Both strains required the highest requirement estimate of digestible Lys to optimize feed conversion.

A trial was conducted to determine the effects of different rearing feed regimens on plasma hormone and metabolite levels and hepatic lipid metabolism and gene expression on sexually mature broiler breeders. Cobb 500 birds were divided into 2 groups at 4 wk and fed either an everyday (ED) or skip-a-day (SKP) regimen. At 24 wk of age, all birds were switched over to an ED regimen. At 26.4 wk, breeder hens were randomly selected and killed at intervals after feeding. Livers were sampled from 4 hens at 4-h intervals for 24 h for a total of 28 samples per treatment. Blood was sampled from 4 hens per sampling time; sampling times were 0, 30, and 60 min and 2 and 4 h after feeding and then every 4 h up to 24 h for a total of 36 samples per treatment. Main feeding regimen, time, and interaction effects were analyzed. Significant interaction effects were found between time and feeding regimen for acetyl-coenzyme A carboxylase and malic enzyme mRNA expression. The peak for acetyl-coenzyme A carboxylase expression was higher in ED-reared birds, whereas the peak for malic enzyme expression was higher in SKP-reared birds. Overall, plasma levels of insulin-like growth factor-II were higher in SKP-reared birds. Overall, plasma corticosterone levels were also higher in SKP-reared birds and significant interaction effects between time and feeding regimen were seen. The expression of apolipoprotein A1 was significantly higher in ED-reared birds: significant interaction effects were also noted. Other researchers also found some of the differences observed in the present study in 16-wk-old pullets. In summary, different feeding regimens alter metabolic responses, some of which carry over into sexual maturity.

This study investigated the effects of fasting and refeeding on AMP-activated protein kinase (AMPK) and carbohydrate response element binding protein (ChREBP) mRNA, protein and activity levels; as well as the expression of lipogenic genes involved in regulating lipid synthesis in broiler chicken (Gallus gallus) liver. Fasting for 24 or 48 h produced significant declines in plasma glucose (at 24 h), insulin and thyroid hormone (T3) levels that were accompanied by changes in mRNA expression levels of hepatic lipogenic genes. The mRNA levels of malic enzyme (ME), ATP-citrate lyase (ACL), acetyl-CoA carboxylase alpha (ACCalpha), fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1) and thyroid hormone responsive Spot 14 (Spot 14) declined in response to fasting. Refeeding for 24 h increased mRNA levels for each of these genes, characterized by a significant increase ('overshoot') above fed control values. No change in mRNA expression of the two AMPK alpha subunit genes was observed in response to fasting or refeeding. In contrast, ChREBP and sterol regulatory element binding protein-1 (SREBP-1) mRNA levels decreased during fasting and increased with refeeding. Phosphorylation of AMPK alpha subunits increased modestly after a 48 h fast. However, there was no corresponding change in the phosphorylation of ACC, a major downstream target of AMPK. Protein level and DNA-binding activity of ChREBP increased during fasting and declined upon refeeding as measured in whole liver tissue extracts. In general, evidence was found for coordinate transcriptional regulation of lipogenic program genes in broiler chicken liver, but specific regulatory roles for AMPK and ChREBP in that process remain to be further characterized.

An investigation was conducted to study insulin-like growth factor (IGF)-I, IGF-II, insulin, glucagon, leptin, triiodothyronine (T(3)), and thyroxine (T(4)) levels in a chicken population divergently selected for P bioavailability (PBA). There were differences in growth and feed efficiency between the 2 lines. Concentrations of IGF-I, IGF-II, and T(3) were significantly greater in the high PBA line compared with the low PBA line, whereas the reverse was true for glucagon. There were no correlations between IGF-I and II and PBA in either line, suggesting that the line differences may be the result of factors other than PBA. Glucagon and IGF-I have different relationships with feed conversion ratio in the high PBA line compared with the low PBA line. There was a significant correlation between PBA and T(3) in the low line and between PBA and T(4) in the high PBA line. Thyroid hormone levels may be an indirect indicator of PBA in growing chickens. The genes in the thyroid hormone pathway may be key in the identification of genes associated with PBA.

To better understand how the proglucagon system functions in birds, we utilized a molecular cloning strategy to sequence and characterize the chicken proglucagon gene that encodes glucagon, glucagon-like peptide (GLP)-1 and GLP-2. This gene has seven exons and six introns with evidence for an additional (alternate) first exon and two promoter regions. We identified two distinct classes of proglucagon mRNA transcripts (PGA and PGB) produced by alternative splicing at their 3'-ends. These were co-expressed in all tissues examined with pancreas and proventriculus showing the highest levels of each. Although both mRNA classes contained coding sequence for glucagon and GLP-1, class A mRNA lacked that portion of the coding region (CDS) containing GLP-2; whereas, class B mRNA had a larger CDS that included GLP-2. Both classes of mRNA transcripts exhibited two variants, each with a different 5'-end arising from alternate promoter and alternate first exon usage. Fasting and refeeding had no effect on proglucagon mRNA expression despite significant changes in plasma glucagon levels. To investigate potential differences in proglucagon precursor processing among tissues, mRNA expression for two prohormone convertase (PC) genes was analyzed. PC2 mRNA was predominantly expressed in pancreas and proventriculus, whereas PC1/3 mRNA was more highly expressed in duodenum and brain. We also determined mRNA expression of the specific receptor genes for glucagon, GLP-1 and GLP-2 to help define major sites of hormone action. Glucagon receptor mRNA was most highly expressed in liver and abdominal fat, whereas GLP-1 and GLP-2 receptor genes were highly expressed in the gastrointestinal tract, brain, pancreas and abdominal fat. These results offer new insights into structure and function of the chicken proglucagon gene, processing of the precursor proteins produced from it and potential activity sites for proglucagon-derived peptide hormones mediated by their cognate receptors.

A trial was conducted to determine the effects of different feeding regimens on plasma hormone and metabolite levels in 16-wk-old broiler breeder pullets. A flock of 350 Cobb 500 breeder pullets was divided in 2 at 28 d of age and fed either every day (ED, 5 pens of 35 birds) or skip-a-day (SKIP, 5 pens of 35 birds) from 28 to 112 d of age. Total feed intake did not differ between the 2 groups. At 112 d, 52 randomly selected pullets from the larger flock of ED-fed pullets, and 76 from the SKIP-fed pullets were individually caged and fed a meal of 74 g (ED) or 148 g (SKIP). Blood samples were collected from 4 pullets in each group by cardiac puncture at intervals after feeding. Plasma was analyzed for insulin, glucagon, insulin-like growth factor-I and insulin-like growth factor-II, triiodothyronine and thyroxine, corticosterone, leptin, glucose, nonesterified fatty acids, triglycerides, and uric acid. Feed retention in the crop was also noted at each interval. In ED birds, the crop was empty by 12 h and in SKIP birds, the crop was empty by 24 h after feeding. The physiological responses to fasting, such as increased glucagon and corticosterone and reduced plasma triglyceride, occurred at times coincidental with crop emptying in both ED and SKIP birds. Overall, mean insulin-like growth factor-I levels were higher (P < 0.05) in ED birds. Triiodothyronine was higher (P = 0.09) in SKIP birds. Overall mean plasma corticosterone was 2-fold higher in SKIP-fed birds, which may be related to the increased length of fasting periods, hunger, and stress. Plasma leptin was consistently higher in ED-fed birds, which was indicative of their more consistent food supply and more stable energy status. In summary, the experiment reported here shows that different feeding regimens can alter hormone and metabolite profiles, in spite of total feed intakes being equal.

Components of the insulin-like growth factor (IGF) system were investigated in chondrocytes isolated from the avian growth plate. The genes for IGF-I, IGF-II, type 1 IGF receptor (IGF-R), IGF binding protein-2 (IGFBP-2), IGFBP-3, IGFBP-5 and IGFBP-7 were found to be expressed in both proliferative and hypertrophic chondrocytes. The expression of IGF-II in proliferative chondrocytes was extremely high relative to IGF-I. Although IGF-I expression was significantly increased in hypertrophic chondrocytes, the level was still low relative to IGF-II. In cell culture, IGF-I stimulated proteoglycan synthesis and increased the expression of Indian hedgehog (Ihh) and type X collagen, markers of chondrocyte differentiation. IGF-II was found to be equally efficacious in stimulating proteoglycan biosynthesis. These observations suggest that IGF-II may play a significant role in avian growth plate physiology, which is consistent with several reports on mammalian endochondral bone growth.

Developmental hormonal changes in Cobb 500 chick embryos and hatched chicks were determined by measuring plasma insulin, glucagon, insulin-like growth factor (IGF)-I, IGF-II, triiodothyronine, thyroxine, and glucose concentrations at different ages of embryogenesis and posthatch development. Plasma samples were obtained daily from 10 d of embryogenesis (10E) through 13 d posthatch and also at 17 and 21 d posthatch. A significant increase in plasma insulin was observed with increasing age from 10E to hatch. Plasma glucagon levels remained low until 17E, and then significantly increased approximately 3-fold at hatch, which corresponded with increasing plasma glucose levels during late embryo development. The plasma insulin to glucagon molar ratio of incubation from 14E to 17E ranged from 2 to 4, and was significantly higher than at any other time during incubation. These results indicate that insulin may be an important promoter of chick embryonic growth by the anabolic drive to promote protein deposition. Insulin and glucagon increased after hatch, which may be due to increased feed consumption and increased utilization of carbohydrates as the key energy source, compared with nutrients obtained through lipolysis and proteolysis in the embryos. Plasma triiodothyronine increased 4-fold from 18E to 20E, and thyroxine increased 3-fold from 16E to 19E. Insulin-like growth factor-I and IGF-II peaked at 14E. Insulin-like growth factor-I steadily increased above embryonic levels during the 3 wk of the posthatch period, whereas IGF-II levels steadily declined. These results suggest that IGF-II may be a more important functionary for chick embryonic development than IGF-I, and that IGF-I may be more important than IGF-II after hatch. The profile of metabolic hormones in the present study may help support an understanding of significant changes that occur in embryonic development and posthatch growth in chicks.

The current study is a comprehensive genome analysis to detect QTL affecting metabolic traits in chickens. Two unique F(2) crosses generated from a commercial broiler male line and 2 genetically distinct inbred lines (Leghorn and Fayoumi) were used in the present study. The plasma glucagon, insulin, lactate, glucose, tri-iodothyronine, thyroxine, insulin-like growth factor I, and insulin-like growth factor II concentrations at 8 wk were measured in the 2 F(2) crosses. Birds were genotyped for 269 microsatellite markers across the entire genome. The program QTL Express was used for QTL detection. Significance levels were obtained using the permutation test. For the 10 traits, a total of 6 and 9 significant QTL were detected at a 1% chromosome-wise significance level, of which 1 and 6 were significant at the 5% genome-wise level for the broiler-Leghorn cross and broiler-Fayoumi cross, respectively. Most QTL for metabolic traits in the present study were detected in Gga 2, 6, 8, 9, 13, and Z for the broiler-Leghorn cross and Gga 1, 2, 4, 7, 8, 13, 17, and E47 for the broiler-Fayoumi cross. Phenotypic variation for each trait explained by all QTL across genome ranged from 2.73 to 14.08% in the broiler-Leghorn cross and from 6.93 to 21.15% in the broiler-Fayoumi cross. Several positional candidate genes within the QTL region for metabolic traits at the 1% chromosome-wise significance level are biologically associated with the regulation of metabolic pathways of insulin, triiodothyronine, and thyroxine.

The 5'-AMP-activated protein kinase (AMPK) plays a key role in regulating cellular energy homeostasis. The AMPK is a heterotrimeric enzyme complex that consists of 1 catalytic (alpha) and 2 regulatory (beta and gamma) subunits. Mutations of the gamma subunit genes are known to affect AMPK functioning. In this study, we characterized the genomic organization and expression of 3 chicken AMPK gamma subunit genes (cPRKAG). Alternative splicing of the second exon of the cPRKAG1 gene resulted in 2 transcript variants that code for predicted proteins of 298 and 276 amino acids. Use of an alternate promoter and alternative splicing of the cPRKAG2 gene resulted in 4 transcript variants that code for predicted proteins of 567, 452, 328, and 158 amino acids. Alternative splicing of exon 3 of the cPRKAG3 gene resulted in the production of "long" and "short" transcript variants that code for predicted proteins of 382 and 378 amino acids, respectively. We found evidence for differential expression of individual gamma subunit gene transcript variants and, in some cases, tissue-specific expression was observed. The cPRKAG subunit genes displayed similar structural features and high sequence homology compared with corresponding mammalian gamma subunit gene homologues.

Three hundred twenty Cobb 500 broiler breeder pullets at 21 wk of age were selected from a flock fed according to Cobb Breeder Management Guide specifications. One hundred sixty pullets at 21 wk of age were switched to ad libitum feeding, and the remaining 160 pullets continued to be control-fed. The pullets were photostimulated at 22 wk and maintained until 36.5 wk. Plasma samples were obtained, BW was determined, and hens were killed for determination of body composition at the following periods: 24 h prior to photostimulation, 2.5 wk after photostimulation, 24 h after first egg, and 36.5 wk following peak egg production. Compared with ad libitum-fed breeders, the restricted breeders had a higher percentage carcass protein and lower percentage carcass fat at all sampling periods. Total egg numbers were greater, and abnormal eggs were less for the restricted pullets compared with the ad libitum-fed pullets at 36.5 wk. Carcass percentage fat of ad libitum-fed pullets was positively related to plasma glucagon, insulin-like growth factor-II (IGF-II), and 17beta-estradiol but negatively related to plasma insulin, insulin/glucagon M ratio, insulin-like growth factor-I (IGF-I), thyroxine (T4), and triiodothyronine (T3). Carcass percentage fat of feed-restricted pullets was negatively related to IGF-I, IGF-II, and T4. The T4 was the most important hormone for predicting the percentage carcass fat in ad libitum-fed pullets, and IGF-I was the most important hormone for predicting the percentage carcass fat in feed-restricted pullets. The percentage carcass protein for ad libitum-fed breeders was positively correlated to IGF-I, T4, T3, insulin/glucagon M ratio, and insulin. Carcass percentage protein for feed-restricted breeders was positively correlated to IGF-I, IGF-II, T4, and glucagon. Stepwise regressions for predicting percentage carcass protein for breeders fed by both systems shows that T3 and IGF-I concentrations were the most important for ad libitum-fed breeders, whereas IGF-II and T4 were best for feed-restricted breeders. The hormone status of breeders may be a key indicator to help predict the body composition and thus support management decisions for maintaining optimum production.

Ghrelin, a peptide hormone produced by the stomach in mammals, stimulates growth hormone release and food intake. Recently, ghrelin was identified and characterized in chicken proventriculus and shown to stimulate growth hormone release but inhibit feed intake. The purpose of this work was to identify and further characterize the ghrelin gene in chickens and in turkeys. Using molecular cloning techniques we have sequenced cDNAs corresponding to chicken (White Leghorn) and turkey ghrelin mRNAs. A total of 844 (chicken) or 869 (turkey) bases including the complete coding regions (CDS), and the 5'- and 3'-untranslated regions (UTRs) were determined. Nucleotide sequence (CDS) predicted a 116 amino acid precursor protein (preproghrelin) for both the chicken and the turkey that demonstrated complete conservation of an N-terminal 'active core' (GSSF) including a serine (position 3 of the mature hormone) known to be a modification (acylation) site important for ghrelin bioactivity. Additional nucleotide sequence was found in the 5'-UTRs of both Leghorn and turkey cDNAs that was not present in broilers or the red jungle fowl. The turkey ghrelin gene, sequenced from genomic DNA templates, contained five exons and four introns, a structure similar to mammalian and chicken ghrelin genes. Ghrelin was highly expressed in proventriculus with much lower levels of expression in other tissues such as pancreas, brain, and intestine. RT-PCR was used to quantify ghrelin mRNA levels relative to 18S rRNA in 3-week-old male broiler chickens. The level of ghrelin mRNA increased in proventriculus in response to fasting but did not decline with subsequent refeeding. Plasma ghrelin levels did not change significantly in response to fasting or refeeding and did not appear to reflect changes in proventriculus ghrelin mRNA levels. Ghrelin mRNA levels declined in broiler pancreas after a 48 h fast and increased upon refeeding. Expression of the gene encoding the receptor for ghrelin (growth hormone secretagogue receptor, GHS-R) and a variant form was detected in a variety of tissues collected from 3-week-old male broiler chickens possibly suggesting autocrine/paracrine effects. These results offer new information about the avian ghrelin and ghrelin receptor genes and the potential role that this system might play in regulating feed intake and energy balance in poultry.

We designed three experiments to determine both the optimal dose of and time on experiment for methimazole (MMI; 1-methyl-2-mercaptimidazole). Our goals were to determine if chicken growth was related to thyroid hormone levels and if intermediary metabolism changed along with changes in thyroid hormone levels. Initiating MMI at one week of age decreased (P<0.01) plasma thyroid levels and growth in four-week old birds. In contrast, initiating MMI at two and three weeks of age decreased (P<0.05) hormone levels without affecting growth as severely. Although initiating MMI at two weeks of age depressed (P<0.05) plasma thyroid hormones at four weeks, there was little change in vitro lipogenesis at four weeks. Again, initiating MMI at one week of age decreased body weight, plasma thyroid hormones and in vitro lipogenesis at four weeks of age. In addition, this treatment also decreased (P<0.05) malic enzyme activity at this same age period. The second experiment showed that MMI, initiated at 14 days, had no significant effect on 28-day body weight and again decreased both plasma T(3) and T(4) but T(3) replacement increased plasma T(3) in both 14-28-day treatment groups. All body weights were similar at 30 days, however. Lastly, diets containing graded levels of MMI decreased thyroid hormones and body weight (0>0.25>0.5>1 g MMI/kg). In contrast, only the two higher levels (0.5 and 1 g MMI/kg) decreased in vitro lipogenesis. Growth depression, caused by MMI feeding, can occur without changes in lipid metabolism. The length of MMI administration may be as important as dose level in obtaining effects (growth, thyroid hormone depression and inhibition of lipogenesis).

In mammals, AMP-activated protein kinase (AMPK) is involved in the regulation of cellular energy homeostasis and, on the whole animal level, in regulating energy balance and food intake. Because the chicken is a valuable experimental animal model and considering that a first draft of the chicken genome sequence has recently been completed, we were interested in verifying the genetic basis for the LKB1/AMPK pathway in chickens. We identified distinct gene homologues for AMPK alpha, beta and gamma subunits and for LKB1, MO25 and STRAD. Analysis of gene expression by RT-PCR showed that liver, brain, kidney, spleen, pancreas, duodenum, abdominal fat and hypothalamus from 3 wk-old broiler chickens preferentially expressed AMPK alpha-1, beta-2 and gamma-1 subunit isoforms. Heart predominantly expressed alpha-2, beta-2 and gamma-1, whereas skeletal muscle expressed alpha-2, beta-2 and gamma-3 preferentially. Moreover, the AMPK gamma-3 gene was only expressed in heart and skeletal muscle. Genes encoding LKB1, MO25 alpha, MO25 beta, and STRAD beta were expressed in all examined tissues, whereas STRAD alpha was expressed exclusively in brain, hypothalamus, heart and skeletal muscle. Alterations in energy status (fasting and refeeding) produced little significant change in AMPK subunit gene transcription. We also determined the level of phosphorylated (active) AMPK in different tissues and in different states of energy balance. Immunocytochemical analysis of the chicken hypothalamus showed that activated AMPK was present in hypothalamic nuclei involved in regulation of food intake and energy balance. Together, these findings suggest a functional LKB1/AMPK pathway exists in chickens similar to that observed in mammals.

This study investigated changes in bone integrity and circulating concentrations of insulin-like growth factor-I (IGF-I) of hens subjected to 2 distinct molting regimens and fed pre- and postmolt diets high in n-3 or n-6 fatty acids. A dual-energy x-ray absorptiometer determined bone mineral density (BMD) of the tibia and humerus of 45 live hens from 62 to 76 wk of age. Densitometric scans were also conducted in excised tibia and humerus at 66, 71, and 76 wk of age. Concentrations of IGF-I were monitored using an homologous RIA at the same ages. The molting treatments consisted of 10 d of fasting + cracked corn for 7 d + pullet developer diet for 10 d or a nonfasting molt (wheat-middlings-based diet for 27 d). Five weeks prior to and after either molt treatment, birds were fed 1 of 2 diets containing dietary n-6/ n-3 fatty acids ratios of 0.6 or 8.0. At the end of the molt (71 wk of age), tibial BMD decreased 30% in fasted and 11% in nonfasted molt regimens, and the fatty acid content of the premolt diet had no effect on the decline in BMD. The BMD of the humerus also decreased during molt with the exception of hens subjected to a nonfasted molt and fed n-3 fatty acid diets in which their BMD values were similar to or greater (at 73 wk of age) than those of controls during the entire experimental period (treatment by bone by age, P < or = 0.0001). Induced molt affected circulating IGF-I concentrations (treatment by age interaction, P < or = 0.0001), and the response was the same regardless of molt regimen (fasting vs. nonfasting) or diet (n-3 vs. n-6 fatty acids). A decrease in IGF-I 54 h postmolt was noted; however, from 13 to 43 d postmolt, all molted birds had elevated IGF-I as compared with controls. In conclusion, a nonfasted molt as compared with fasted molt was less detrimental to bone mineralization; dietary n-6/n-3 fatty acid ratios in the pre- and postmolt diets had little effect on the decline of skeletal integrity during molt, and circulating IGF-I concentrations were affected by molt.

Molecular genetic selection on individual genes is a promising method to genetically improve economically important traits in chickens. A resource population was developed to study the genetics of growth, body composition, skeletal integrity, and metabolism traits. Broiler sires were crossed to dams of 2 diverse, highly inbred lines (Leghorn and Fayoumi), and the F1 birds were intermated by dam line to produce broiler-Leghorn and broiler-Fayoumi F2 offspring. Growth, body composition, skeletal integrity, and hormonal and metabolic factors were measured in 713 F2 individuals. Insulin-like growth factor-I (IGF1) was selected for study as a biological and positional candidate gene. A single nucleotide polymorphism (SNP) was identified between the founder lines in the IGF1 promoter region, and a PCR-RFLP assay was developed. A mixed model was used to statistically analyze associations of IGF1-SNP1 with phenotypic traits. The IGF1-SNP1 had significant associations with most recorded traits, except metabolic traits. Strong interactions between the IGF1 gene and genetic background on growth traits in the 2 F2 populations suggest that genetic interaction is an important aspect for consideration before using the IGF1-SNP1 in marker-assisted selection programs. Several beneficial effects (improved growth, increased breast muscle weight, decreased abdominal fat, and enhanced skeletal integrity) associated with 1 allele indicate the presence of 1 or more loci near IGF1-SNP1 controlling biologically diverse and economically important traits in chickens.

Although changes in dietary protein levels change metabolism in the broiler chicken, there is little information concerning the time course of the process of adaptation. Therefore, male Hubbard broiler chickens were fed diets containing either 12 or 30% crude protein from 7 to 28 d of age and then were switched to the opposite level for an additional 9 d. Birds were bled and killed at 0, 2, 5, 7and 9 d following the reversals. Data taken at these intervals included those involved in vitro lipogenesis (IVL), growth and feed consumption, hepatic enzyme activities and plasma hormones and metabolites. Birds fed the lower level of crude protein were smaller in growth from 7 to 28 d. Feeding these birds a higher protein diet from 28 to 37 d improved both growth and feed efficiency in comparison to controls. Lipogenesis was also greater and plasma insulin-like growth factor-I (IGF-I) less in birds fed the lower protein diet. Switching dietary treatments increased and decreased lipogenesis as birds were switched from high to low and low to high protein diets, respectively. Half-maximal changes were observed 2 d after the reversal and maximal changes 5 d after the reversal. In contrast, switching dietary treatments decreased and increased plasma IGF-I as birds were switched from high to low and low to high protein diets, respectively. Half-maximal changes were observed 2 d after the reversal. Of the three hepatic enzymes monitored, malic enzyme activity most closely followed the rapid changes in IVL. In the present study, plasma IGF-I may be a more sensitive indicator of changes in dietary protein than changes in intermediary metabolism.

Leptin is an adipose and liver tissue-derived secreted protein in chickens that has been implicated in the regulation of food intake and whole-body energy balance. In this study, the metabolic clearance and tissue uptake of leptin were examined in the chicken (Gallus gallus). Four-week-old broiler males were infused with (125)I-labeled mouse leptin. Chromatography of radiolabeled leptin in plasma produced two peaks, one at 16 kDa (free leptin) and a free iodine peak. No leptin binding protein in blood was detected. Leptin was cleared with a half-life estimate of 23 min. In order to investigate the tissue distribution and uptake of radiolabeled leptin, multiple tissues were removed from infused birds at 15 and 240 min post-infusion, and trichloroacetic acid (TCA)-precipitable radioactivity was determined. The amounts of radioactivity at 15 min post-infusion in the tissues in rank order were: kidney, testis, lung, spleen, heart, liver, small and large intestine, gizzard, pancreas, bursa, leg and breast muscle, adrenals, and brain. A slightly different pattern of distribution was observed at 240 min post-infusion. We conclude from these studies that unlike mammals, no circulating leptin binding protein is present in chickens. Leptin is metabolized and cleared very rapidly from blood by the kidney.

We showed previously that Met deficiency at 0.25% of the diet causes elevations in plasma triiodothyronine (T3) in broilers. In the present study, plasma levels of thyroid hormones as well as insulin-like growth factors (IGF)-I and -II were measured in chicks fed 3 deficient levels of total Met. Control (0.5%) and Met-deficient diets (0.4, 0.3, and 0.2%) were fed to male broilers from 8 to 22 d of age. Additional groups of control chicks were pair-fed with the Met-deficient ones. Chicks receiving 0.4% Met increased feed intake by 10% with no significant change in body weight. The more severe Met deficiencies of 0.3 and 0.2% caused graded reductions in feed intake and weight gain. However, corresponding pair-fed control chicks were significantly heavier. These changes suggest more marked alterations in metabolic processes with 0.3 and 0.2% Met than with 0.4% Met. Liver weights were heavier in chicks fed 0.3 and 0.2% Met but not 0.4%. Plasma T3 was higher in all deficient chicks compared with the free-fed control, which was significant only with 0.3% Met. However, with 0.3 and 0.2% Met, plasma T3 was significantly elevated compared to pair-fed controls. Plasma thyroxine (T4) was lower in all deficient groups, which was significant only with 0.2% Met, whereas no significant differences occurred between deficient chicks and their pair-fed controls. Plasma IGF-I levels were not significantly different, but they were consistently lower in deficient chicks and deserve further study. Plasma IGF-II was significantly less in chicks fed 0.2% Met compared to pair-fed controls suggesting that Met deficiency interferes with IGF-II metabolism. We concluded that a deficit of dietary Met altered plasma T3 and IGF-II levels, but the effect was dependent on the degree of deficiency.

Seven-day-old chickens were fed diets containing 18% crude protein + 0 or 1g methimazole/kg to produce either euthyroid or hypothyroid groups of birds at 28 days of age. These two groups were then offered diets containing either 0 or 1mg triiodothyronine (T(3))/kg diet. Birds were sampled at 0, 2, 5, and 8 days following the onset of the T(3) treatment. Measurements taken at these intervals included in vitro hepatic lipogenesis (IVL), growth and feed consumption, hepatic enzyme activities (malic enzyme, ME; isocitrate dehydrogenase, ICD; and aspartate amino transferase, AAT), plasma hormones (T(3); thyroxine, T(4); insulin like growth factors I, IGF-I; and insulin like growth factors II, IGF-II) and metabolites (glucose; fatty acids, NEFA; triglyerides; uric acid). Hypothyroidism decreased IVL and ME at 28 days of age; however, T(3) supplementation for 2 days restored both IVL and ME. Paradoxically, continuing T(3) replenishment for an additional 3-6 days decreased IVL without affecting ME activity. In contrast, supplemental T(3) decreased IVL in euthyroid birds, regardless of the dosing interval, but had no effect on ME activity. Methimazole decreased plasma T(3), T(4), uric acid, and IGF-I, but did not affect IGF-II at 28 days. Giving T(3) to birds previously on methimazole increased plasma IGF-I as did feeding a control diet. Supplemental T(3) increased NEFA in both euthyroid and hypothyroid birds, but only for a short period following the initiation of supplementation (2 days post-supplementation). These data may help to explain some of the apparent reported dichotomies in lipid metabolism elicited by changes in the thyroid state of animals. In addition, most metabolic changes in response to feeding T(3) occurred within 2-5 days, suggesting that changes in intermediary metabolism preceded morphological changes. In conclusion, the thyroid state of the animal will determine responses to exogenous T(3)., (Copyright 2002 Elsevier Science Inc.)

Broiler breeder pullets were divided into two groups at 21 wk of age. One group was given free access to feed (ad libitum) and the other fed a limited amount of feed (restricted). At 22 wk, all birds were photostimulated and maintained throughout an egg-laying cycle ending at 36 wk. Samples of liver and abdominal fat pad were collected just before photostimulation (prelight), after photostimulation at first egg and at peak egg production (plateau). Hepatic expression of sterol regulatory element binding protein-1, ATP-citrate lyase, fatty acid synthase, malic enzyme, acetyl-CoA carboxylase and stearoyl-CoA (Delta9) desaturase 1 genes in ad libitum birds declined from their highest levels just before photostimulation as the birds came into and maintained egg production. In contrast, the restricted birds had significant (P < 0.05) increases in the expression of these genes after photostimulation at first egg with a subsequent decline as they reached peak egg production. Hepatic expression of fatty acid binding protein, VLDL apolipoprotein (apoVLDL-II) and apoB genes increased significantly (P < 0.05) in both ad libitum and restricted breeders after photostimulation, whereas apoA1 gene expression declined during this time. Abdominal fat pad weights were significantly (P < 0.05) higher in the ad libitum compared with restricted birds after photostimulation. Lipoprotein lipase in this tissue showed a pattern of expression similar to that observed for the hepatic lipogenic enzyme genes. In conclusion, feed restriction during the pullet-to-breeder transition period significantly (P < 0.05) altered hepatic lipogenic gene expression in broiler breeders.

The bi-guanide metformin is used to treat noninsulin dependent diabetes in obese patients. In addition to having antihyperglycemic effects, metformin is also anorectic and reduces BW. These studies were performed to determine if metformin possesses similar properties in chickens. Metformin-HCl was administered to 14-day-old broiler chickens at either 300 or 600 mg/kg per day in the drinking water for 10 d while monitoring BW and feed intake. No changes in water intake were observed, while feed intake and daily gains were only significantly reduced by the 600 mg/kg dose. After oral administration of a single dose of 300 mg/kg metformin-HCl, feed intake was significantly reduced by 4 h and remained suppressed for greater than 24 h relative to controls. Plasma hormones and metabolites (glucose, lactate, insulin, glucagon, uric acid, nonesterified fatty acid, and triglycerides) were monitored at 1, 2, 3, 6, and 24 h posttreatment. Significant and acute decreases in blood glucose, insulin, and triglycerides were observed at 3 h posttreatment as compared to controls. Opposing acute increases in glucagon and NEFA levels were also observed at 3 h followed by an increase in uric add 6 h posttreatment. These observations suggest that metformin induces metabolic changes in birds, similar to that observed in mammals and may act in a common manner. Metformin-HCl may be useful in glucose metabolism studies by inducing hypoglycemia, a condition rarely observed in birds.

An avian uncoupling protein (UCP) gene homolog was recently sequenced from skeletal muscle and was proposed to have a role in thermogenesis in chickens, ducks and hummingbirds. Since mammalian UCP 2 and UCP 3 also appear to have functions associated with energy and substrate partitioning and body weight regulation, the purpose of this study was to further characterize chicken UCP under conditions of nutritional stress and/or leptin administration. Male 3-week-old chickens were starved for 24 or 48 h and then half of each group was refed for an additional 24 h. In a follow-up experiment, chickens were fed or starved for 48 h with or without leptin administration. Feed deprivation increased UCP mRNA expression in skeletal muscle by up to 260% (P<0.001), and in a time-dependent manner in pectoralis muscle. Refeeding for 24 h normalized muscle UCP mRNA levels. Leptin administration had no effect on muscle UCP. Chicken muscle UCP mRNA levels were highly correlated with plasma triglyceride and non-esterified fatty acid (NEFA) concentrations, and with circulating levels of insulin, insulin-like growth factor (IGF)-I and IGF-II. These results suggest that, as in mammals, avian UCP is up-regulated during feed deprivation and is highly correlated with increased fatty acid oxidation and flux into skeletal muscle.

Thermal conditioning of chicks results in improvements in performance and thermotolerance at marketing age. Conditioning has been found to be a sensitive process, dependent on age and the temperature used. The objective of this study was to assess the optimal timing and temperature for the conditioning processes. Six separate trials were conducted on male broiler chickens: the first two aimed to find the optimal age for thermal conditioning (1 to 5 d of age); the other four evaluated the optimal thermal conditioning temperature between 36 and 40.5 C. At 42 d of age chickens were thermally challenged to evaluate their ability to cope with acute heat stress. The highest body weight was achieved when thermal conditioning had been applied at the age of 3 d, and it coincided with low feed intake and higher to significantly higher feed efficiency. These treated chickens showed relatively lower mortality rate under thermal challenge and lower to significantly lower Triiodothyronine (T3) concentration in Trial 2. Chicks that had been thermally conditioned at ambient temperatures (Ta) of 36 and 37.5 C at the age of 3 d demonstrated the best performance characteristics and the ability to reduce T3 concentration to the lowest levels during thermal challenge. It can be suggested, therefore, that a Ta between 36.0 and 37.5 C, applied at 3 d of age is optimum for thermal conditioning of broiler chickens.

The polypeptide hormone leptin is produced by both adipose tissue and the liver and has been shown to induce satiety in chickens. In this study we have investigated the developmental regulation of leptin mRNA expression in growing broiler chickens. Leptin expression generally increases in all tissues from 1-12 weeks of age. In the subcutaneous fat depot there is an apparent pattern of increased leptin mRNA expression occurring at 2, 6, and 10 weeks post-hatch. This pattern was not evident in the other tissues surveyed and may relate to the cycle of loading and unloading of adipocytes with lipid. No consistent gender differences in leptin expression patterns were detected in the tissues surveyed, as is often observed in mammals. Positive correlations between metabolic body weight and adipose leptin expression levels were observed. Leptin expression by the liver was highly correlated with metabolic body weight from 1-6 weeks of age, and uncorrelated from 6-12 weeks of age. This pattern of increasing liver leptin expression with increasing body weight during the early rapid growth phase of the bird may be due to limited fat storage during this period, which is followed by rapid body fat accumulation from 6-12 weeks. The characterization and tissue specific distribution of leptin mRNA expression in the growing broiler indicate similar patterns of leptin production to that of growing mammals. Leptin may be involved in lipid flux through the adipocyte as well as the shift in lipid metabolism to increased storage during pre-puberty.

In contrast to most vertebrates, GH reportedly has no effect upon somatic growth of the chicken. However, previous studies employed only one to two dosages of the hormone, and limited evidence exists of a hyperthyroid response that may confound its anabolic potential. This study evaluated the effects of 0, 10, 50, 100 and 200 microgram/kg body weight per day chicken GH (cGH) (0-200 GH) infused i.v. for 7 days in a pulsatile pattern to immature, growing broiler chickens (9-10 birds/dosage). Comprehensive profiles of thyroid hormone metabolism and measures of somatic growth were obtained. Overall (average) body weight gain was reduced 25% by GH, with a curvilinear, dose-dependent decrease in skeletal (breast) muscle mass that was maximal (12%) at 100 GH. This profile mirrored GH dose-dependent decreases in hepatic type III deiodinase (DIII) activity and increases in plasma tri-iodothyronine (T(3)), with bot! h also maximal (74 and 108% respectively) at 100 GH. No effect on type I deiodinase was observed. At the maximally effective dosage, hepatic DIII gene expression was reduced 44% versus controls. Despite dose-dependent, fold-increases in hepatic IGF-I protein content, circulating IGF-I was not altered with GH infusion, suggesting impairment of hepatic IGF-I release. Significant, GH dose-dependent increases in plasma non-esterified fatty acid and glucose, and overall decreases in triacylglycerides were also observed. At 200 GH, feed intake was significantly reduced (19%; P<0.05) versus controls; however, additional control birds pair-fed to this level did not exhibit any responses observed for GH-treated birds. The results of this study support a pathway by which GH impacts on thyroid hormone metabolism beginning at a pretranslational level, with reduced hepatic DIII gene expression, translating to reduced protein (enzyme) ex! pression, and reflected in a reduced level of peripheral T(3)-degrading activity. This contributes to decreased conversion of T(3) to its inactive form, thereby elevating circulating T(3) levels. The hyper-T(3) state leads to reduced net skeletal muscle deposition, and may impair release of GH-enhanced, hepatic IGF-I. In conclusion, GH has significant biological effects in the chicken, but profound metabolic actions predominate that may confound positive, IGF-I-mediated skeletal muscle growth.

There are conflicting results concerning the role of the thyroid hormones in lipid metabolism. The experiments in this report were designed to examine the role of T(3) in modifying responses obtained by shifting birds from moderate to low protein diets. Birds were grown from 7 to 28 d on a diet containing 18% protein. At this time, birds were switched to a diet containing 12% protein +/- T(3) The switch was accomplished either immediately or after a 24 hr fast. Measurements taken included in vitro lipogenesis (IVL), hepatic enzyme activities and plasma metabolites and thyroid hormones. Simply switching to birds to the low protein diet increased IVL, but rates were similar for three days following the switch. Feeding T(3) in this same regimen resulted in lower, but again, constant rates of IVL. In contrast, although switching protein levels after a 24 hr fast increased IVL, the rate after two days of refeeding was nearly double that following one day. This accentuated response was somewhat attenuated by including T(3) in the diet. Neither fasting nor refeeding altered plasma T(3) relative to ad libitum values. Supplemental dietary T(3) increased plasma T(3) and results were not affected by feeding regimens. Plasma T(4) was greatest in birds fasted for 24 hr and least in birds fed T(3) suggesting that feeding regimens may regulate the conversion of T(4) to T(3) It is suggested from this study that some of the effects of alterations in dietary feeding regimens can be modulated by T(3)

The effect of intracerebroventricular (i.c.v.) injection of leptin was investigated using broiler and Single Comb White Leghorn (SCWL)-type chickens. These represent relatively fast- and slow-growing birds, respectively. The i.c.v. injection of leptin decreased food intake in both broilers and Leghorns in a dose-dependent manner. The most efficacious dose appeared to be 10 microg in both types of chickens. Water intake was generally not affected by leptin, indicating that this effect was not due to general malaise. It appears that leptin can act within the central nervous sytstem of birds to decrease food intake.

We determined the effect of dietary protein on the distribution of insulin-like growth factor (IGF) binding proteins in chicken plasma. Three groups of male broilers (n = 6 per group) were fed (ad libitum) isocaloric diets containing 12, 21 or 30% dietary protein. Birds were fed respective diets beginning at 7 days of age and killed at 28 days. No differences were observed between adequate (21%) and high (30%) protein intakes for any of the parameters investigated (growth criteria, plasma levels of IGF-I, growth hormone or IGF-binding proteins). Feeding protein deficient diets (12%) resulted in a 34% decrease in body weight, 17% decrease in feed intake and a 39% increase in feed/gain ratio. IGF-binding proteins in plasma samples were separated by SDS-PAGE and transferred to nitrocellulose sheets. Nitrocellulose blots were probed with [125I]chicken IGF-II. Four regions of binding activity corresponding to 70, 43, 30 and 24 kDa were observed in all samples. Birds consuming 12% dietary group protein had less than 50% of the 43-kDa binding activity of birds consuming 21 or 30% dietary protein. The 30-kDa binding activity was 42% lower in the 12% dietary protein group compared to birds consuming adequate protein. In contrast, 70- and 24-kDa binding activities were not influenced by dietary protein. Chickens consuming 12% dietary protein had higher levels of growth hormone and lower levels of IGF-I than those consuming 21 or 30% dietary protein. These data indicate that in chickens, the circulating levels of at least two independent IGF-binding proteins are influenced by dietary protein.

Despite well documented anabolic effects of GH in mammals, a clear demonstration of such responses in domestic poultry is lacking. Recently, comprehensive dose-response studies of GH have been conducted in broilers during late post-hatch development (8 to 9 weeks of age). GH reduced feed intake (FI) and body weight gain in a dose-dependent manner, whereas birds pair-fed to the level of voluntary FI of GH-infused birds did not differ from controls. The reduction in voluntary FI may involve centrally mediated mechanisms, as hypothalamic neuropeptide Y protein and mRNA were reduced with GH, coincident with the maximal depression in FI. Growth of breast muscle was also reduced in a dose-dependent manner. Circulating IGF-I was not enhanced by GH, despite evidence that early events in the GH signaling pathway were intact. A GH dose-dependent increase in circulating 3,3',5-triiodothyronine(T3) paralleled decreases in hepatic 5D-III monodeiodinase activity, whereas 5'D-I activity was not altered. This confirms that a marked hyperthyroid response to GH occurs in late posthatch chickens, resulting from a decrease in the degradative pathway of T3 metabolism. This secondary hyperthyroidism would account for the decreased skeletal muscle mass (52) and lack of enhanced IGF-I (53) in GH-treated birds. Based upon these studies, it is now evident that GH does in fact have significant effects in poultry, but metabolic responses may confound the anabolic potential of the hormone.

The purpose of this experiment was to study the effects of dietary protein on metabolic adaptations in birds given triiodothyronine (T3) at 28 d of age. Knowledge about the role of dietary protein in thyroid metabolism is lacking. Male broiler chickens were fed diets containing either 120 or 300 g crude protein/kg from 7 to 28 d. At this time, one-half of each group was given that diet supplemented with 1 mg/kg T3. Birds were sampled at 0, 2, 5, 7, 9 and 12 d following the initiation of the T3 treatments. Measurements taken at these intervals included in vitro lipogenesis (IVL), growth and feed consumption, hepatic enzyme activities and plasma thyroid hormones and metabolites. As expected, IVL was greater at 28 d in birds fed the lower level of protein. Moreover, when T3 was added to either diet, IVL decreased by 50% after 5 d. Plasma IGF-I and T4 were greater while T3 and GH were less when birds were fed the higher level of crude protein. Plasma T3 increased and T4 decreased 3 d following the addition of T3 to diets containing either level of crude protein. Most metabolic changes in response to feeding T3 occurred within 2 to 5 d, suggesting that changes in intermediary metabolism preceded morphological changes that we have previously seen. In addition, dietary protein levels may not affect adaptive responses to T3.

An experiment was conducted to study the interrelationships between dietary fat and protein levels in the regulation of lipid metabolism in the broiler chicken. Birds were fed diets containing 300, 600, or 1,200 kcal ME from fat (corn oil) with either 124 or 190 g CP/kg. Two additional experimental diets contained 234 or 285 g CP and 300 kcal ME from fat. Regardless of fat level, birds fed the diets containing 124 g CP/kg weighed less and were less efficient than birds fed diets containing 190 g CP/kg. The diet containing 600 kcal as fat decreased lipogenesis and malic enzyme activity (P < 0.05) in birds fed the diet containing 190 g CP/kg diet, but not in birds fed the diet containing 124 g CP/kg. Birds fed the latter level of protein required at least 1,200 kcal as fat to express any significant decrease in lipogenesis or malic enzyme activity (P < 0.05). Dietary fat did not affect plasma levels of triiodothyronine (T3), thyroxine (T4), or insulin-like growth factor-I (IGF-I). Feeding diets containing 124 g CP/kg resulted in decreased plasma T4 and IGF-I and elevated T3 (P < 0.05). Increasing dietary protein (compared to increasing dietary fat) increased body weights, IGF-I, T4 and decreased lipogenesis, malic enzyme activity, and T3. Both of these regimens involve decreasing dietary carbohydrate at equal rates, but results differed. Although replacement of dietary carbohydrates with either fat or protein reduce precursors for fat synthesis, both energy sources have additional unique effects on metabolism. Dietary protein levels modulate metabolic effects of dietary fat.

Previous research has reported that elevations in circulating growth hormone (GH) levels in meat-type chickens depresses feed intake (FI) more than 30%. It is known that the product of the obese gene, leptin, functions to regulate FI and energy expenditure. To investigate the effect of GH on leptin gene expression, broiler chickens were infused with recombinant chicken GH. To separate any secondary effects of a GH-induced reduction in FI on leptin expression, groups of birds were pair-fed to an average level of voluntary intake similar to GH-treated birds, but received no GH treatment. GH treatment induced a dose-dependent increase in liver leptin gene expression, as measured by reverse transcriptase-polymerase chain reaction, whereas leptin expression in adipose tissue was unchanged. Conversely, in chickens pair-fed (feed-restricted) there was a decrease in leptin gene expression in both tissues. These results provide evidence of a direct effect of GH on leptin gene expression, which is independent of any effects on intake attributable to GH-treatment, and suggest differential regulation of leptin expression between adipose tissue and liver. The results of these experiments provide the first evidence of a relationship between GH and leptin in domestic birds.

Previous studies in avian species have reported time-dependent losses in muscle glycogen with prolonged feed withdrawal (FW). However, cervical dislocation was used to collect tissues, a method that results in significant involuntary muscle convulsions. In this study, cervical dislocation alone was found to reduce muscle glycogen by 23%, therefore, barbiturate overdose was used to collect tissue samples before and after FW, at the end of refeeding, and from continuously fed controls at each interval. Additionally, plasma samples from 6-wk-old male chickens were taken at the initiation and end of a 24-hr feed withdrawal, and at various times during refeeding. After 24 hr of FW, liver glycogen decreased markedly (77%; P < 0.05), whereas muscle glycogen decreased slightly and transiently, such that it returned to and remained at control levels, even after prolonged (72 hr) FW. Plasma glucose was decreased, whereas glucagon was elevated after a 24-hr feed withdrawal (P < 0.05), when compared with control concentrations. Muscle glycogen levels were not significantly increased over control levels after refeeding, but liver glycogen was increased by 380% (P < 0.05). Feed deprivation followed by refeeding resulted in increased circulating insulin and glucose levels when compared with control levels. Therefore, by using methods of tissue collection that ensure that muscle glycogen determinations are not confounded by artifactual degradation, these results verify that regulation of avian muscle glycogen stores is similar to that in mammals.

Leptin, the polypeptide hormone encoded by the obese gene, is secreted by adipose tissue and has been shown to induce satiety and increase energy expenditure in mammals. In this study, we confirmed the presence of a leptin homolog in liver and adipose tissues of broiler chickens. Leptin expression was also detected in chicken embryonic liver and yolk sac. The effects of hormone treatment on leptin expression in chickens were also investigated. Leptin expression in the liver is increased by insulin and dexamethasone and decreased by glucagon and estrogen. There was no induction of leptin expression in adipose tissue by any treatment, whereas only estrogen decreased adipose expression. The differential effect on liver and adipose tissue suggests that adipocytes in chickens may be expressing leptin at a maximal rate or that its mechanism of expression regulation differs from liver. The localization of leptin expression and tissue-specific effects of hormone treatments on leptin expression observed in chickens may indicate a relationship between leptin and avian lipid metabolism.

The development of a homologous RIA for chicken insulin-like growth factor-II (cIGF-II) and its application to investigate the developmental changes in IGF-II in the chicken and turkey are described. A double-antibody RIA has been developed using recombinantly derived cIGF-II as antigen, radiolabelled tracer and standard. Serial dilutions of chicken and turkey plasma were parallel to serial dilutions of cIGF-II standard. We have also established that acid/ethanol extraction of chicken and turkey plasma reduced possible interference of insulin-like growth factor-binding proteins in the RIA. Consumption of a low-protein diet by male chickens lowered plasma IGF-I twofold, whereas IGF-II levels were unchanged. Food withdrawal evoked an increase in circulating IGF-II, while IGF-I levels were reduced. Refeeding returned both growth factors to normal circulating concentrations. During chick embryo incubation, plasma IGF-II levels were tenfold higher than those of IGF-I. In the turkey embryo, plasma IGF-II concentrations were higher than those of IGF-I. During the post-hatch period. IGF-II levels declined with age in chickens. In the growing turkey, IGF-II levels were consistently higher than IGF-I levels. The application of the homologous RIA to monitor plasma levels during embryonic development and post-hatch growth in avian species will provide more accurate comparisons of results from studies on the role of IGF-II in growth and metabolism of domestic birds.

The efficacy of exogenous IGFs to stimulate growth and modulate protein and fat deposition was examined in a number of broiler chicken lines. From around 600 g body weight the chickens received a continuous infusion of vehicle (0.1 M acetic acid), human recombinant IGF-I or [Gly1]IGF-II at 300 microg/kg body weight per day, or a combined infusion of 150 microg/kg/day of each IGF for 2 weeks. Experiment 1 used commercial broiler female chickens and included measurements of nitrogen balance, Ntau-methylhistidine excretion and muscle protein synthesis rates. In Experiment 2 the same treatments were applied to three experimental lines of chickens selected for high food consumption (relatively fat), high food utilisation efficiency (relatively lean), or at random (control). IGF-I, but not IGF-II, significantly increased growth rate and food utilisation efficiency by around 10-15% in each experiment, an effect which was consistent across all genotypes. Nitrogen balance was significantly increased by IGF-I in Experiment 1 as was carcass nitrogen content in Experiment 2, indicating that the increased growth was in lean tissue. Carcass fat was consistently reduced in chickens receiving IGF-I and was related to the levels of circulating IGF-I (r2 = 0.30, P < 0.01) but not triiodothyronine. Protein synthesis rates were unaffected by treatment and could not account for increased growth rate. However, there was a significant reduction in Ntau-methylhistidine excretion indicating a reduced rate of muscle protein breakdown in IGF-I-treated chickens (1. 56%/day vs 2.05%/day for IGF-I-treated vs controls, P < 0.05). The efficiency of feed utilisation was inversely related to the rate of protein breakdown (r2 = 0.25, P < 0.01). In conclusion, these experiments are the first to report an enhancement of growth and food utilisation efficiency by broiler chickens receiving exogenous IGF-I. Our results show that IGF-I may be important in controlling the growth and efficiency of food utilisation of young chickens at least in part by modulating the rates of protein breakdown., (Copyright 1998 Academic Press.)

Indian River male broiler chickens growing from 7 to 28 d of age were fed on diets containing either 120 or 210 g crude protein and 0 or 1 mg triiodothyronine (T3)/kg diet to study in vitro lipogenesis (IVL). In addition, a carry-over period (180 g crude protein/kg diet from 28 to 40 d of age) was used to test the persistence of prior treatment effects. The higher protein level increased, but T3 decreased (P < 0.01) growth and feed consumption at 28 d of age. The lower protein level increased (P < 0.05) and T3 decreased IVL in 28-d-old chickens. These effects were only sustained for 6 d following the switch to a common diet at 28 d. IVL at 40 d of age was not affected by either crude protein or T3 fed during the 7-28 d period. The higher protein level increased plasma insulin-like growth factor-1 during the period from 7 to 28 d; however, this effect lasted for only 6 d following the switch to a common diet. Plasma growth hormone (GH) at 28 d of age was inversely related to dietary protein level. Changing to a common level of crude protein did not change plasma GH values at 12 d, indicating that the nutritional state of the young chicken may affect GH at a later period of life. Metabolic changes noted in this study were rapid and maintained for a short period of time following the feeding of a common diet.

Insulin-like growth factor-I (IGF-I) effects on chicken growth and development are poorly understood. This study examined the effect of IGF-I on protein synthesis rates in various tissues in the male broiler chicken. At three weeks of age, osmotic minipumps were subcutaneously implanted in the scapular area. Chickens were infused with either chicken IGF-I (450 micrograms/kg BW/day) or saline. After treatment for 5 days, the chickens received a flooding dose of [3H]-phenylalanine, and were sacrificed 20 min later. Wing vein blood samples were taken at 0, 5, 10 and 20 min post-injection. The following tissues were removed and frozen for analysis: pectoralis muscle, gastrocnemius muscle, heart, liver, and small intestine. In vivo total protein synthesis measurements were made using the double-label technique. Contractile protein degradation was evaluated using intracellular free 3-methylhistidine concentrations in skeletal muscle. There were no significant differences in absolute or relative body growth rates over the treatment period. Skeletal muscle (pectoralis and gastrocnemius) weights were significantly decreased with IGF-I treatment, while heart weight was significantly increased. Plasma insulin levels were significantly lower in IGF-treated chickens compared to that in control birds. There was no effect of IGF-I on protein synthesis rates in any of the tissues examined. Intracellular free 3-methylhistidine concentrations were higher in both the gastrocnemius (17%) and pectoralis muscles (25%) of chickens treated with IGF-I. This data demonstrates that IGF-I may have an indirect effect to regulate muscle protein turnover rates.

A large amount of research, primarily in mammals, has defined to a great extent the pleiotropic effects of the IGF system on growth, development, and intermediary metabolism. Similar elucidations in poultry were hindered to some extent by the absence of native peptides (IGF-I and IGF-II) until their purification, followed by the production of recombinant chicken IGFs. In many ways IGF physiology in birds is similar to that in other species, including but not limited to the fact that IGF-I synthesis is both GH- and GH-independent, and that autocrine-paracrine IGF action is evident. However, it is clear that several unique differences in IGF physiology exist between birds and mammals. For example, more IGF is present in the free form in chickens, and the biological responses to the IGFs is different in several metabolic pathways in birds compared to mammals. To date, no unique IGF-II receptor has been identified in birds. Despite an increasing understanding of the IGFs in aves, several important questions remain to be answered. What is the role of IGF-II in embryo development and posthatch growth? Does an IGF-II receptor entity exist in nonmammalian species? How does nutrition affect IGF-I and IGF-II gene expression, and can this information be used to enhance poultry production? What is the biochemical composition of the IGFBPs, and what are their roles in birds? Can the genetic variation present in poultry be used to positively modify IGF gene expression and physiology? How do the IGFs regulate intermediary metabolism? What is the role of the IGFs in the etiology of several disease states associated with rapid growth in poultry, including tibial dyschondroplasia, obesity, ascites, and spiking mortality syndrome? Answers to these questions are relevant to our understanding of the basic mechanisms of IGF physiology as well as possibly assisting in the amelioration of problems found in modern poultry production.

Insulin-like growth factor-I (IGF-I) mediates many of the effects of growth hormone (GH). The regulation of IGF-I, independent of GH, is methodologically difficult to assess in vivo, as hypophysectomy results in derangement of many pituitary hormone axes in addition to GH, and a gene knockout model is not available. The recessive sex-linked dwarfing (SLD) gene (dw) in chickens results in a lack of functional target tissue GH receptors due to a variety of molecular defects, which provides a unique model for evaluating GH-independent regulation of IGF-I. In the present study, the impact of 3,3', 5-triiodo-l-thyronine (T3) on circulating and tissue IGF-I was determined in normal versus SLD birds. Adult, nonovulatory female normal and SLD chickens were restrict-fed 40 g of feed/kg bw/day containing 0, 0.5, or 1.0 ppm T3, resulting in supplementation levels of 0 (control), 20 (low dose), or 40 (high dose) microg T3/kg bw/day for 10 days. Samples of GH target tissues including liver, abdominal fat pad, skeletal muscle (pectoralis major), and spleen were extracted and assayed for IGF-I. Plasma T3, T4, GH, and IGF-I were determined by homologous RIA. Tissue GH binding was determined for hepatic membranes by radioreceptor assay. Under control conditions, dwarf chickens were markedly hypersomatotropic (33.3 +/- 4.1 ng GH/ml plasma; mean +/- SEM) compared to normals (2.4 +/- 3.9 ng/ml), and T3 supplementation reduced this to normal levels. Despite the high circulating level of GH in dwarfs, plasma IGF-I was low compared to normal controls (dwarfs 1.5 +/- .9 ng/ml; normals 5. 3 +/- .9 ng/ml; P = 0.004), but this difference was eliminated with low-dose T3. In this study, tissue IGF-I was undetectable in liver and pectoralis muscle in adults (55 weeks of age) of both genotypes, under all treatments. In contrast, adipose tissue IGF-I was relatively high and did not differ (P = 0.84) between genotypes under control conditions (normals 776.5 +/- 236.7; dwarfs 844.6 +/- 236.7 pg/mg protein), but was increased in normals and decreased in dwarfs, resulting in higher levels (P = 0.02) in the normal (1249.9 +/- 200.0 pg/mg protein) than in the dwarf genotype (558.4 +/- 200.0 pg/mg protein) at the higher level of T3 supplementation. This relationship was somewhat reversed in spleen, where T3 tended to decrease tissue IGF-I concentration in normals and increase it in dwarfs. The low level of plasma IGF-I despite nonmeasureable hepatic IGF-I tissue concentrations suggests that IGF-I synthesis by extrahepatic tissues contributes to the circulating pool of IGF-I. The relatively high control levels of adipose tissue IGF-I in the dwarf genotype further suggest that considerable IGF-I synthesis exists that is GH-independent in this extrahepatic tissue. The presence of GH action, however, may mediate the effects of other hormones that can influence local IGF-I production in this tissue, as reflected by the differential response to T3 supplementation between genotypes. The tissue-specific nature of the effect of T3 on IGF-I production supports an additional point of regulation of hormone action at the target tissue level.

Although changes in dietary crude protein levels change metabolism in broiler chickens, there is little information concerning the time course of the process of adaptation. Therefore, male Indian River broiler chickens were fed diets containing either 120 or 210 g protein/kg from 7 to 28 d of age and then were fed the other level for an additional 12 d. Birds were bled and killed at 0, 2, 5, 7, 9 and 12 d following the reversals. Measurements taken at these intervals included in vitro lipogenesis, growth and feed consumption, hepatic enzyme activities and plasma metabolites and metabolites. Birds fed the lower level of crude protein were smaller and less efficient in growth from 7 to 28 d. Feeding these birds a higher protein diet from 28 to 40 d improved both growth and feed efficiency. Lipogenesis was also greater and plasma insulin-like growth factor-I (IGF-I) less in birds fed the lower protein diet. Switching dietary treatments increased and decreased lipogenesis as birds were switched from high to low protein and from low to high protein diets, respectively. Half-maximal changes were observed 4 d after the reversal and maximal changes 7 d after the reversal. In contrast, switching dietary treatments decreased and increased plasma IGF-I as birds were switched from high to low protein and from low to high protein diets, respectively. Half-maximal changes were observed 2 d after the reversal. Of the three hepatic enzymes monitored, malic enzyme activity most closely followed the rapid changes in in vitro lipogenesis. Plasma IGF-I may be a more sensitive indicator of changes in dietary protein than changes in intermediary metabolisms.

The metabolic clearance of chicken IGF-I (cIGF-I), cIGF-II, human IGF-I (hIGF-I), and hIGF-II was examined in the chicken using 125I-labelled growth factors. Superose-12 chromatography of plasma collected at 7.5 min post-infusion revealed peaks of radioactivity corresponding to 150 and 43 kDa and unbound tracer. Statistical analysis of trichloroacetic acid (TCA)-precipitable radioactivity in sequential plasma samples as well as following chromatography of the same samples revealed that clearance of the radiolabelled peptides followed an apparent triphasic pattern. The close similarity of the individual chromatographically defined pools in their clearance rate compared with the three components described by TCA precipitation strongly suggested their identity. Both free 125I-labelled cIGF-II (3.11 min) and hIGF-II (3.01 min) were cleared at a greater rate than their IGF-I counterparts. Unbound hIGF-I was cleared at a greater rate than cIGF-I (4.45 vs 5.66 min respectively). A similar pattern for clearance was evident in the radio-labelled growth factors associated with the 43 kDa component, although at a longer half-life. There was no difference in the apparent clearance of the radiolabelled growth factors associated with the 150 kDa component between IGF-I or -II or between species. Analysis of the chromatographic profiles of radioactive IGF-I peptides complexed to serum proteins versus those bound to labelled IGF-II peptides revealed the presence of a large molecular mass binding protein in vivo. Ligand blotting of chicken serum determined that a binding protein with a mass of 70 kDa was detectable with 125I-IGF-II probes only, and was not present in pig serum. In addition, tissue uptake of 125I-cIGF-I and -II was evaluated. Similar patterns of tissue distribution and uptake were observed for 125I-cIGF-I and -II, except that cIGF-II uptake by the liver exceeded that of 125I-cIGF-I at 15 min post-infusion. The rank order of tissue distribution was as follows: kidney > testis > heart > liver > pancreas > small intestine > cartilage > bursa > gizzard > leg muscle > breast muscle > brain. We conclude from these studies that the clearance of IGFs from the compartments identified in blood and the potential target tissues is dependent on their interactions with IGF-binding proteins and receptors.

A homologous RIA for the determination of toxic gizzerosine activity in commercial fish meals has been developed. Three polyclonal antibodies (GR316, GR415, and GR418) were produced in female rabbits and extensively characterized for their potential use in individual RIA. The RIA had lower detection limits of 0.048, 0.78, and 0.39 ng/mg using the three respective antibodies. Because gizzerosine is derived from lysine and histidine, crossreactivity with these amino acids, and with histamine was examined. The antibodies crossreacted with histamine from 21 to 100%. No crossreactivity with histidine or lysine was observed for any of the three antibodies. Antibody GR415 was chosen for determination of gizzerosine in extracted fish meal samples because crossreactivity of histamine using this antibody was only present at high concentrations, and the Ka value for gizzerosine was 10-fold greater than for histamine. A mild buffer extraction procedure was used, resulting in 98% gizzerosine recovery. Displacement curves from extracted and serially diluted fish meal samples were parallel with gizzerosine standard. Inter-and intra-assay coefficients of variation were 11 and 15%, respectively. We used the RIA for determination of gizzerosine activity in a pool of 23 fish meal samples of known gizzerosine scores (determined with a chick bioassay), and histamine content. The partial correlation coefficient between gizzerosine content determined by the RIA and gizzerosine scores from the bioassay was high (0.83), and significant (P < 0.01). There were also significant correlations between gizzerosine scores and histamine content of the fish meals (0.63, P < 0.01), and between histamine content and gizzerosine levels determined by the RIA (0.59, P < 0.01). The application of the homologous RIA for the determination of gizzerosine activity in commercial fish meals could be of importance for the prevention of gizzard erosions in the poultry industry, and for studying gizzerosine-induced pathology and metabolism.

Indian River male broiler chickens growing from 7 to 30 d of age were fed on diets containing crude protein levels ranging from 120 to 300 g/kg plus 0 or 1 mg triiodothyronine (T3)/kg diet. The purpose of this study was to examine the effects of these treatments on lipogenesis after a common diet was fed (180 g crude protein/kg diet from 30 to 56 d of age). Dietary treatment groups were sampled at 30 and 56 d. In vitro lipogenesis was determined by incubating liver explants for 2 h at 37 degrees in Hanks' salts containing 25 mM-HEPES and 10 mM-[2-14C]acetate and then measuring acetate incorporation into total lipid. Growth and feed consumption from 7 to 30 d increased (P < 0.01) as dietary protein increased from 120 to 210 g/kg diet. Both measurements decreased as crude protein increased from 210 to 300 g/kg diet. T3 decreased (P < 0.01) growth and feed intake during this period. Low-protein (< 180 g/kg) diets increased (P < 0.05) and T3 decreased lipogenesis in 30-d-old chickens. Although birds given T3 from 7 to 30 d grew at the greatest rate from 30 to 56 d of age, the final body weight was still less than controls. In vitro lipogenesis at 56 d of age was not affected by either of the two dietary treatments. In contrast, the relative size of the abdominal fat pad (g/kg body weight) at 56 d was decreased by feeding T3 from 7 to 30 d. Any changes in metabolism elicited by either dietary protein levels or hormone treatments may be specific to the particular dosing interval and are not sustained when a common diet is fed during a repletion period.

The development of a homologous radioimmunoassay (RIA) for chicken insulin-like growth factor-I (cIGF-I) and its use to investigate the developmental changes in IGF-I in the chicken and turkey is described. A double-antibody RIA has been developed using recombinantly derived cIGF-I as antigen, radiolabelled tracer and standard. The resulting immunoassay has a minimum detection limit of 0.035 ng and effective dose of 2.5 ng. Dose-response curves of chicken and turkey plasma and tissue extracts were parallel with cIGF-I standard. The antiserum is specific for IGF-I as no cross-reactivity with chicken IGF-II, insulin, glucagon, gastrin or avian pancreatic polypeptide was observed. We have also established that acid/ethanol extraction of chicken and turkey plasma reduced possible interference of IGF-binding proteins (IGFBPs) in the RIA. Comparison of IGF-I immunoactivity in unextracted and acid/ethanol-extracted samples following gel filtration under acidic and neutral conditions indicates that the cIGFBPs may be acid-labile. Analyses of samples from growing chickens and turkeys using the homologous avian reagents revealed higher IGF-I concentrations than if the IGF were quantified using heterologous mammalian-derived reagents. A similar pattern was observed when tissue extracts were assayed for IGF-I content. The application of the homologous RIA to monitor blood and tissue IGF-I levels during embryonic development and posthatch growth in avian species will provide more accurate comparisons of results from studies on the role of IGF-I in growth and metabolism of domestic birds.

Male broiler chickens growing from 7 to 35 d were fed on a diet containing 150 g crude protein (N x 6.25)/kg diet supplemented with lysine to equal that in diets containing 166, 183 and 200 g crude protein/kg diet (Expt 1). A second group of male broiler chickens growing over the same period were fed on a diet containing 120 g crude protein/kg supplemented with lysine, arginine, tryptophan, threonine and isoleucine equal to that in diets containing 144, 172 and 200 g crude protein/kg diet (Expt 2). Growth was improved by lysine supplementation but not to the level attained by feeding 200 g crude protein/kg (Expt 1). Lysine, arginine, tryptophan, threonine and isoleucine supplementation of a low-protein diet also improved growth, but growth again fell short of that attained by feeding a diet containing 200 g crude protein/kg. Plasma insulin-like growth factor-1 and thyroxine concentrations increased and triiodothyronine decreased as the crude protein level increased from 150 to 200 g/kg diet. Supplemental lysine did not affect plasma levels of these hormones. Although dietary crude protein levels noticeably changed rates of in vitro lipogenesis, changing either the level of a single limiting amino acid or the levels of several limiting amino acids did not change lipogenesis.