HWS Lines Research Articles

Fed and fasted chicks from lines divergently selected for low or high body weight have differential hypothalamic appetite-associated factor mRNA expression profiles

We have demonstrated that chicken lines which have undergone intense divergent selection for either low (LWS) or high (HWS) body weight (anorexic and obese containing, respectively) have differential food intake threshold responses to a range of intracerebroventricular injected neurotransmitters. The study reported herein was designed to measure endogenous appetite-associated factor mRNA profiles between these lines in an effort to further understand the molecular mechanisms involved in their differential eating patterns. Whole hypothalamus was collected from 5 day-old chicks that had been fasted for 180min or had free access to food. Total RNA was isolated, reverse transcribed, and real-time PCR performed. Although mRNAs encoding orexigenic neuropeptides including agouti-related peptide, neuropeptide Y (NPY), prolactin-releasing peptide, and visfatin did not differ in expression between the lines, NPY receptor 5 mRNA was greater in fed LWS than HWS chicks, but fasting decreased the magnitude of difference. Anorexigenic factors including amylin, corticotropin releasing factor (CRF) and ghrelin were not differentially expressed between lines, while mRNA abundance of calcitonin, CRF receptor 1, leptin receptor, neuropeptide S, melanocortin receptor 3, and oxytocin were greater in LWS than HWS chicks. Pro-opiomelanocortin mRNA was lower in LWS than HWS chicks, while fasting decreased its expression in both lines. These results suggest that there are differences in gene expression of appetite-associated factors between LWS and HWS lines that might be associated with their differential food intake and thus contribute to differences in severity of anorexia, body weight, adiposity, and development of obesity.

Expression of carnitine palmitoyl-CoA transferase-1B is influenced by a cis-acting eQTL in two chicken lines selected for high and low body weight

Carnitine palmitoyl-CoA transferase-1B is a mitochondrial enzyme in the fatty acid oxidation pathway. In a previous study, CPT1B was identified as differentially expressed in the hypothalamus of two lines of chickens established by long-term selection for high (HWS) or low (LWS) body weight. Mammals have three paralogs (CPT1a, b and c) while nonmammalian vertebrates only have two (CPT1A, B). CPT1A is expressed in liver and CPT1B in muscle. CPT1c is expressed in hypothalamus, where it regulates feeding and energy expenditure. We identified an intronic length polymorphism, fixed for different alleles in the two populations, and mapped the hitherto missing CPT1B locus in the chicken genome assembly, to the distal tip of chromosome 1p. Based on molecular phylogeny and gene synteny we suggest that chicken CPT1B is pro-orthologous of the mammalian CPT1c. Chicken CPT1B was differentially expressed in both muscle and hypothalamus but in opposite directions: higher levels in hypothalamus but lower levels in muscle in the HWS than in the LWS line. Using an advanced intercross population of the lines, we found CPT1B expression to be influenced by a cis-acting expression quantitative trait locus in muscle. The increased expression in hypothalamus and reduced expression in muscle is consistent with an increased food intake in the HWS line and at the same time reduced fatty acid oxidation in muscle yielding a net accumulation of energy intake and storage. The altered expression of CPT1B in hypothalamus and peripheral tissue is likely to be a mechanism contributing to the remarkable difference between lines.

Neuropeptide Y is associated with changes in appetite-associated hypothalamic nuclei but not food intake in a hypophagic avian model

While neuropeptide Y (NPY) has been studied extensively per its pronounced role in food intake stimulation as well as its role in central pathways governing eating disorders, it has to our knowledge not been studied in polygenic models of hypo- and hyperphagia. Thus, the present study was designed to measure central NPY-associated food intake in lines of chickens that have undergone long-term genetic selection for low (LWS) or high (HWS) body weight and exhibit hypo- and hyperphagia, respectively. LWS chicks did not respond with any magnitude of altered food intake to any dose of NPY tested, while HWS chicks responded to all doses of NPY at similar magnitudes throughout the duration of observation. Both lines responded with similar increases in c-Fos immunoreactivity in the lateral hypothalamus and both divisions of the paraventricular nucleus; there were no significant line or line by treatment interactions. These data support the hypothesis that differences exist in the central NPY system of chicks from LWS and HWS lines and may provide novel insight for understanding NPY control of appetite.

Differentially expressed genes in hypothalamus in relation to genomic regions under selection in two chicken lines resulting from divergent selection for high or low body weight

Long-term divergent selection for low or high body weight from the same founder population has generated two extremely divergent lines of chickens, the high- (HWS) and low-weight (LWS) selected lines. At selection age (56days), the lines differ by more than nine times in body weight. The HWS line chickens are compulsive feeders, whereas in the LWS line, some individuals are anorexic and others have very low appetite. Previous studies have implicated the central nervous system and particularly the hypothalamus in these behavioural differences. Here, we compared the mRNA expression in hypothalamus tissue from chickens on day4 post-hatch using oligonucleotide arrays and found that the divergent selection had resulted in minor but multiple expression differences. Differentially expressed genes were enriched in processes 'DNA metabolism, repair, induction of apoptosis and metabolism'. Several differentially expressed genes participate in the regulation of neuronal plasticity and development, including apoptosis, or are neurotransmittor receptor subtypes. Less change was seen when comparing hypothalamic neuropeptide mediators of appetite such as the melanocortin receptors. The genomic locations of these differentially expressed genes were then compared to the locations of growth QTLs and to a genome-wide map of chromosomal regions that have been under divergent selection between the lines. The results indicate which differentially expressed hypothalamic genes have responded to the divergent selection and that the results predict that it is more likely to find causative genes among these most differentially expressed genes. Because of such differential gene expression in hypothalamus, the lines may adapt behaviourally different particularly to the post-hatch situation when independent feeding to obtain energy is established.

AICAR and Compound C regulate food intake independently of AMP-activated protein kinase in lines of chickens selected for high or low body weight

AMP-activated protein kinase (AMPK) functions to maintain cellular and body energy balance. Our aim was to investigate the effect of intracerebroventricular (ICV) administration of AMPK stimulator AICAR and AMPK inhibitor Compound C on food intake in lines of chickens that had undergone long-term selection from a common founder population for high (HWS) or low (LWS) body weight. AICAR caused a quadratic dose-dependent decrease in food intake in LWS but not HWS chicks. Compound C caused a quadratic dose-dependent increase in food intake in HWS but not in LWS chicks. Key aspects of the AMPK pathway, including upstream kinases mRNA expression, AMPK subunit α mRNA expression and phosphorylation, and a downstream target acetyl CoA carboxylase (ACC) phosphorylation were not affected by either AICAR or Compound C in either line. The exception was a significant inhibitory effect of AICAR on ACC phosphorylation ratio due to increased total ACC protein content without changing phosphorylated ACC protein levels. Thus, the anorexigenic effect of AICAR in LWS chicks and orexigenic effect of Compound C in HWS chicks resulted from activation or inhibition of other kinase pathways separate from AMPK. These results suggest genetic variation in feeding response for central AICAR and Compound C in chickens, which may contribute to the different body weights between the HWS and LWS lines.

Genetic diversity and population structure in lines of chickens divergently selected for high and low 8-week body weight

A long-term selection experiment for high or low 8-wk BW in White Plymouth Rock chickens was conducted to study effects of selection on BW and correlated characters. Two lines [high (HWS), low (LWS) weight] were established and have undergone 48 generations of selection. The lines were managed to curtail inbreeding and to maintain similar population structures; such is necessary for equitable comparison of selection response between lines. Our objective was to test the success of that breeding strategy by characterizing genetic diversity and inbreeding in these lines. A pedigree of 5,998 individuals was assembled, with 68 founders, 2,962 HWS chickens, and 2,968 LWS chickens. Inbreeding coefficients (F) were calculated for each line. Maximum F was 0.53 and 0.61, mean F was 0.26 (SD 0.15) and 0.30 (SD 0.17), and change in F was 1.3 and 1.6% per generation in LWS and HWS lines, respectively. The effective population size was 38.3 in LWS and 32.1 in HWS lines. The effective number of founders was 15.7 in both lines, and the effective number of ancestors was 17.5 and 15.5 in LWS and HWS lines, respectively. Thirty ancestors accounted for 90% of the genetic makeup of both lines. Seven male and eight female founders still contributed to both lines at generation 48, although some contributed more to one line than the other. Family sizes were similar for males and females of each line, with males having larger family sizes with greater variance. Accumulated inbreeding was high and effective population size was low, as expected in closed lines. Effective number of founders was relatively low compared with actual number of founders, indicating some contributed more than others to the last generation. Family size statistics indicated that fewer males than females were used, leading to the observed levels of inbreeding. Given their similarity in genetic diversity and family size, it can be concluded that breeding decisions throughout the project resulted in similar population structures in the lines.

Proviral integrations and expression of endogenous Avian leucosis virus during long term selection for high and low body weight in two chicken lines

BackgroundLong-term selection (> 45 generations) for low or high juvenile body weight from a common founder population of White Plymouth Rock chickens has generated two extremely divergent lines, the LWS and HWS lines. In addition to a > 9-fold difference between lines for the selected trait, large behavioural and metabolic differences between the two lines evolved during the course of the selection. We recently compared gene expression in brain tissue from birds representing these lines using a global cDNA array analysis and the results showed multiple but small expression differences in protein coding genes. The main differentially expressed transcripts were endogenous retroviral sequences identified as avian leucosis virus subgroup-E (ALVE).ResultsIn this work we confirm the differential ALVE expression and analysed expression and number of proviral integrations in the two parental lines as well as in F9 individuals from an advanced intercross of the lines. Correlation analysis between expression, proviral integrations and body weight showed that high ALVE levels in the LWS line were inherited and that more ALVE integrations were detected in LWS than HWS birds.ConclusionWe conclude that only a few of the integrations contribute to the high expression levels seen in the LWS line and that high ALVE expression was significantly correlated with lower body weights for the females but not males. The conserved correlation between high expression and low body weight in females after 9 generations of intercrosses, indicated that ALVE loci conferring high expression directly affects growth or are very closely linked to loci regulating growth.

Extremely Different Behaviours in High and Low Body Weight Lines of Chicken are Associated with Differential Expression of Genes Involved in Neuronal Plasticity

Long-term selection (> 45 generations) for low or high body weight from the same founder population has generated two extremely divergent lines of chickens, the low (LWS) and high weight (HWS) lines, which at the age of selection (56 days) differs by more than nine-fold in body weight. The HWS line chickens are compulsive feeders, whereas, in the LWS line, some individuals are anorexic and others have very low appetites. The involvement of the central nervous system in these behavioural differences has been experimentally supported. We compared a brain region at 0 and 56 days of age containing the major metabolic regulatory regions, including the hypothalamus and brainstem, using a global cDNA array expression analysis. The results obtained show that the long-term selection has produced minor but multiple expression differences. Genes that regulate neuronal plasticity, such as actin filament polymerisation and brain-derived neurotrophic factor, were identified as being differentially expressed. Genes involved in lipid metabolism were over-represented among differentially expressed genes. The expression data confirm that neural systems regulating feeding behaviours in these lines are different. The results suggest that the lines are set in separate developmental trajectories equipped with slightly different nervous systems. We suggest that the lines adapt behaviourally different to changing situations post hatch, such as the transition from dependence on yolk to feeding, in order to obtain energy. The present study has identified and exemplifies the kind of changes that may underlie the extreme differences in such behaviours.

Differential Appetite‐Related Responses to Central Neuropeptide S in Lines of Chickens Divergently Selected for Low or High Body Weight

The anorexigenic 20 amino acid neuropeptide S (NPS) has not been studied in an animal model of hypo- or hyperphagia. The present study aimed to elucidate whether central NPS appetite-related effects are different in lines of chickens that had undergone long-term divergent selection for low (LWS) or high (HWS) body weight and that were hypo- and hyperphagic, respectively. It took a longer time for food intake to be reduced in LWS than HWS chicks administered the lowest dose of NPS tested (0.14 nmol) and, at the highest dose tested (0.56 nmol), they had a greater reduction in food intake than did HWS chicks. HWS chicks responded with a similar magnitude of food intake reduction that was independent of NPS dose. Although water intake was reduced concurrently with food intake after central NPS in both lines, blood glucose concentrations were not affected. Hypothalamic signalling was different between the lines. Although both lines respond to central NPS with decreased c-Fos immunoreactivity in the lateral hypothalamus, the periventricular nucleus had increased c-Fos immunoreactivity in LWS but not HWS chicks. After central NPS treatment, there was increased c-Fos immunoreactivity in the paraventricular nucleus in HWS but not LWS chicks. These data support the notion of differences in the central NPS system between the LWS and HWS lines and infer that central NPS may differentially affect appetite-related processes in other species that contain hypo- and hyperphagic individuals.

Differential feeding responses to central alpha-melanocyte stimulating hormone in genetically low and high body weight selected lines of chickens

This study was conducted to compare the effects of central α-MSH, a potent anorexigenic signal, in lines of chickens that have undergone long-term divergent selection for low (LWS) or high (HWS) body weight. Chicks from both lines were centrally injected with 0, 24, 120 or 600 pmol α-MSH and feed and water intake were concurrently measured thereafter for a total of 180 min. The LWS line responded to all doses of α-MSH with a similar potent decrease in feed intake at all observation times. The HWS line only responded to 600 pmol α-MSH with decreased feed intake. α-MSH did not influence water intake in either line. To determine if differential hypothalamic signaling was associated with the anorexigenic effect, c-Fos immunoreactivity was measured in appetite-related hypothalamic nuclei after 600 pmol central α-MSH injections. c-Fos immunoreactivity was increased in the dorsomedial hypothalamus, paraventricular nucleus (PVN) and ventromedial hypothalamus in both lines after α-MSH; however, the magnitude of increase was greater in LWS than in HWS chicks at the PVN (136% vs. 47% increase over controls, respectively). Based on behavior observations, the number of feeding and exploratory pecks is decreased with greater magnitude after α-MSH in the LWS line. Additionally, α-MSH was associated with increased deep rest in both lines, and may be a secondary effect to reduced ingestion. These data support that the LWS line has a lower threshold for the anorexigenic effect of central α-MSH while in the HWS line this threshold is higher, and that this difference may be associated with differential hypothalamic signaling. Genetic variation exists in the threshold of anorexigenic response for central α-MSH in LWS and HWS lines of chickens with possible implications to other species including humans.

Central α‐melanocyte stimulating hormone causes more potent anorexigenic effects in a line of chickens containing anorexic individuals as compared to a line that is obese

α‐melanocyte stimulating hormone (α‐MSH) causes potent anorexigenic effects in both mammals and birds when centrally administered. We evaluated the central effects of α‐MSH in lines of chickens selected for low (LWS) or high (HWS) body weight for 49 consecutive generations. LWS contains some anorexic individuals while the HWS chickens are hyperphagic. Central α‐MSH more potently reduced feed intake in LWS than HWS. In a second experiment, we found that central α‐MSH reduced exploratory pecking in LWS but not HWS chicks. α‐MSH more potently reduced locomotion and increased deep rest in the LWS than HWS line. Although feed intake was reduced in LWS, their feeding efficiency was increased by central α‐MSH. In conclusion, LWS and HWS lines of chickens responded differently to central α‐MSH; the central melanocortin system may contribute to their differential body weights.

Differential autonomic nervous system response in obese and anorexic chickens ( Gallus gallus)

Effect of reserpine on body weight (BW), feed intake (FI), brain and plasma catecholamine and indoleamine concentrations in high- (HWS) and low- (LWS) weight selected lines of chickens was investigated. Chicks from each line were assigned to three treatment groups and injected intraperitoneally with 0, 1.25, or 2.50 mg/kg of reserpine at hatch, and again at 5 weeks-of-age. Chick BW and FI were determined weekly. At 7 weeks-of-age, 12 males and females from each group were sacrificed for neurotransmitter analysis. In the HWS line there was a dose-dependent decrease in BW through 7 weeks-of-age, whereas in the LWS line BW decreased only through the first 2 weeks-of-age. In the LWS line, norepinephrine (NE), epinephrine, and 3,4-dihydroxyphenylacetate concentrations decreased in the brain in a linear and quadratic manner in response to reserpine, but not in the HWS line. Both lines showed linear decreases in dopamine levels in response to reserpine; however, serotonin was not affected by reserpine. Chickens in the HWS line had greater plasma NE, and lower 5-hydroxyindoleacetic acid than those in the LWS line. In conclusion, it appears that chickens from the HWS line were more sensitive to the BW reducing effects of reserpine than those from the LWS line, with the latter appearing to have greater sympathetic nervous system activity.

Leptin effects on food and water intake in lines of chickens selected for high or low body weight

There is an association between autonomic nervous system output and obesity. The sympathetic nervous system stimulates lipid metabolism and regulates food intake and, hence, body weight. Leptin, produced by adipocytes in proportion to their size, has been shown to directly stimulate the satiety center. In the experiment reported here, food and water intake were compared after intracerebroventricular administration of human recombinant leptin to lines of chickens that had undergone divergent selection for over 45 generations from a common White Rock base population for high (HWS) or low (LWS) body weight at 8 weeks-of-age. Leptin caused a linear decrease in food intake in chickens from the LWS line whereas no effect was observed in those from the HWS line. The HWS chickens tended to have reduced water intake post leptin administration. Others reported that leptin decreased food intake in both broiler and Leghorn chickens. Leptin concentration in the central nervous system may not contribute directly to the difference of body weight between HWS and LWS chickens.

HWS line in action

HWS line in action

Selection for Body Weight at Eight Weeks of Age: 11. Ovulation and Oviposition Patterns

Two experiments were conducted to study oviposition patterns and ovarian activity at various physiological ages in S15 and S16 generation pullets from lines selected bidirectionally for high (HWS) and low (LWS) juvenile body weight. The fixed periods of lay for random samples of pullets from the date of first egg were 20, 40, 60, and 80 days in the first experiment and 40, 80, 120, and 160 days in the second experiment.HWS pullets matured significantly earlier than LWS pullets in both generations. The frequency and the percentage hen-day production (%HDP) of defective eggs were significantly greater in the HWS than LWS line in both generations. Delayed sexual maturity did not significantly change the pattern of defective egg production for a fixed period of lay. In the HWS line the %HDP of defective eggs progressively increased until about 40 days of lay and tended to decline thereafter, while in the LWS line the percentage decreased from 20 to 40 days and then stabilized. The frequency of normal unbroken eggs to 80 days of production did not differ among lines either in the S15 or S16 generation. When measurements were extended to 160 days of lay in the S16 generation HWS pullets produced significantly more normal eggs than LWS pullets. Possible effects of ovarian activity on the egg production pattern during the laying cycle are discussed. There was a significantly higher incidence of internal laying and atrecia of the growing follicles in the HWS than in the LWS pullets. Ovarian activity, evidenced by the number of developing and ruptured follicles, was significantly greater in the HWS than the LWS line, as was the incidence of ova developing in pairs.Product moment correlations and multiple regressions among the measurements of ovarian activity were calculated within lines. In the HWS line, 62.5% of the variation in %HDP of defective eggs could be accounted for by the %HDP of normal eggs and the number of developing follicles while in the LWS line these variables accounted for only 7.29% of the variation in %HDP of defective eggs.