Decades of intensive genetic selection in the domestic chicken (Gallus gallus domesticus) have enabled the remarkable rapid growth of today’s broiler (meat-type) chickens. However, this enhanced growth rate was accompanied by several unfavorable traits (i.e., increased visceral fatness, leg weakness, and disorders of metabolism and reproduction). The present descriptive analysis of the abdominal fat transcriptome aimed to identify functional genes and biological pathways that likely contribute to an extreme difference in visceral fatness of divergently selected broiler chickens.

An F(2) population (695 individuals) was established from broiler chickens divergently selected for either high (HG) or low (LG) growth, and used to localize QTL for developmental changes in body weight (BW), shank length (SL9) and shank diameter (SD9) at 9 weeks. QTL mapping revealed three genome-wide QTL on chromosomes (GGA) 2, 4 and 26 and three suggestive QTL on GGA 1, 3 and 5. Most of the BW QTL individually explained 2-5% of the phenotypic variance. The BW QTL on GGA2 explained about 7% of BW from 3 to 7 weeks of age, while that on GGA4 explained 15% of BW from 5 to 9 weeks. The BW QTL on GGA2 and GGA4 could be associated with early and late growth respectively. The GGA4 QTL also had the largest effect on SL9 and SD9 and explained 7% and 10% of their phenotypic variances respectively. However, when SL9 and SD9 were corrected with BW9, a shank length percent QTL was identified on GGA2. We identified novel QTL and also confirmed previously identified loci in other chicken populations. As the foundation population was established from commercial broiler strains, it is possible that QTL identified in this study could still be segregating in commercial strains.

The avian immune genome -a glass half-

Both thyroid hormone (T3) and growth hormone (GH) are important regulators of somatic growth in birds and mammals. Although T3-mediated gene transcription is well known, the molecular basis of T3 interaction with GH on growth and development of birds remains unknown. In earlier studies, we discovered that exogenous GH alone increased accumulation of visceral fat in young chickens, while the combination of GH injections and dietary T3 worked synergistically to deplete body fat. In the present study, cDNA microarray and quantitative RT-PCR analyses enabled us to examine hepatic gene expression in young chickens after chronic manipulation of thyroid status and GH injection alone or in combination with T3. Thyroid status modulates expression of common and unique sets of genes involved in a wide range of molecular functions (i.e., energy metabolism, storage and transport, signal transduction, protein turnover and drug detoxification). Hepatic expression of 35 genes was altered by hypothyroidism (e.g., ADFP, ANGPTL3, GSTalpha, CAT, PPARG, HMGCL, GHR, IGF1, STAT3, THRSPalpha), whereas hyperthyroidism affected expression of another cluster of 13 genes (e.g., IGFBP1, KHK, LDHB, BAIA2L1, SULT1B, TRIAD3). Several genes were identified which have not been previously ascribed as T3 responsive (e.g., DEFB9, EPS8L2, ARHGAP1, LASS2, INHBC). Exogenous GH altered expression of 17 genes (e.g., CCAR1, CYP2C45, GYS2, ENOB, HK1, FABP1, SQLE, SOCS2, UPG2). The T3+GH treatment depleted the greatest amount of body fat, where 34 differentially expressed genes were unique to this group (e.g., C/EBP, CDC42EP1, SYDE2, PCK2, PIK4CA, TH1L, GPT2, BHMT). The marked reduction in body fat brought about by the T3+GH synergism could involve modulation of hormone signaling via altered activity of the Ras superfamily of molecular switches, which control diverse biological processes. In conclusion, this study provides the first global analysis of endocrine (T3 and GH) regulation of hepatic gene transcription in the chicken.

egg ω-3 content for the fl axseed diet (from 546 to 578 mg/60g egg; P=0.02) and for the Linpro diet (from 415 to 438 mg/60g egg; P=0.07). As well, an overall decrease (P=0.01) in ω-6 to ω-3 ratio (from 1.53 to 1.47) was noted. Enzyme addition increased (P=0.02) the NSP digestibility from 17.2 to 24.6%. There was no signifi cant effect of enzyme addition on total tract fat digestibility. Overall, hens fed the Linpro diets had higher (P=0.02) fat digestibility than those fed the fl axseed diets (95.6 vs 89.9%). It could be concluded from this study that both dietary ingredient (i.e., Linpro) and diet processing along with enzyme supplementation had positive effects on feed utilization and the production of ω-3-enriched eggs.

Excessive adiposity has become a major drawback in meat-type chicken production. However, few studies were conducted to analyze the liver expression of genes involved in pathways and mechanisms leading to adiposity. A previous study performed by differential display on RNAs extracted from chicken livers from lean and fat lines allowed us to isolate cDNA products of genes with putative differential expression. In this study, a cDNA microarray resource was developed from these products together with cDNAs from genes involved in or related to lipid metabolism. This resource was used to analyze gene expression in the liver from lean and fat chickens. Some genes were found with a difference in expression between lean and fat animals and/or correlated to adipose tissue weight. Cytochrome P450 2C45, thought to play a role in biotransformation of steroids and poly-unsaturated fatty acids, was more expressed in lean chickens whereas fatty acid synthase, stearoyl-CoA desaturase, sterol response element binding factor 1 and hepatocyte nuclear factor 4, respectively involved in lipogenesis and its regulation, were more expressed in fat chickens. These results indicate that mechanisms involved in the expression and regulation of lipogenic genes could play a key role in fatness ontogenesis in chickens from lean and fat lines.

Introduction For decades, we have focused on the investigation of a single or, on occasion, multiple endocrine factors that regulate avian growth and development (8, 12). Much of our current understanding of the importance of a functional thyroid axis came from studies where thyroid-active substances were fed to young broiler chickens (12-14, 17). The metabolically-active thyroid hormone, T 3 , plays a major role in maintenance of metabolic rate, body temperature and body composition (12) via paracrine adjustments in the insulin/glucagon (I/G) molar ratio (28). Short-term treatment of pre-market broiler chickens with a low level of dietary T 3 (0.25 ppm) reduces accumulation of excessive body fat and increases accretion of protein in skeletal muscle (12). On the other hand, the incorporation of a goitrogen [propylthiouracil (PTU)] into feed of young chickens leads to hypothyroidism, hyper-secretion of insulin and GH, retarded growth rate and mild obesity (12). The obesity of hypothyroid chickens is largely due to the absence of T 3 negative feedback on GH secretion (23) which in turn contributes to elevated insulin secretion and a higher I/G molar ratio. Early studies in young broiler chickens showed that exogenous cGH fails to increase either growth rate or plasma IGF-I, albeit the accumulation of body fat was increased (7, 12, 15, 37, 39). This is unlike the typical mammalian response to exogenous GH which includes increased growth rate, elevated plasma IGF-I and a leaner body mass (11, 26, 40). Thus, slight obesity, elevated GH, higher I/G molar ratio, and down-regulation of the hepatic GH receptor (GHR) are common features of the GH resistance observed in young chickens (12, 36). The importance of a functional somatotropic axis is clearly revealed in the GHR-deficient dwarf chicken, which has a retarded growth rate, short

The genetic networks that govern the differentiation and growth of major tissues of economic importance in the chicken are largely unknown. Under a functional genomics project, our consortium has generated 30 609 expressed sequence tags (ESTs) and developed several chicken DNA microarrays, which represent the Chicken Metabolic/Somatic (10 K) and Neuroendocrine/Reproductive (8 K) Systems (). One of the major challenges facing functional genomics is the development of mathematical models to reconstruct functional gene networks and regulatory pathways from vast volumes of microarray data. In initial studies with liver-specific microarrays (3.1 K), we have examined gene expression profiles in liver during the peri-hatch transition and during a strong metabolic perturbation — fasting and re-feeding — in divergently selected broiler chickens (fast vs. slow-growth lines). The expression of many genes controlling metabolic pathways is dramatically altered by these perturbations. Our analysis has revealed a large number of clusters of functionally related genes (mainly metabolic enzymes and transcription factors) that control major metabolic pathways. Currently, we are conducting transcriptional profiling studies of multiple tissues during development of two sets of divergently selected broiler chickens (fast vs. slow growing and fat vs. lean lines). Transcriptional profiling across multiple tissues should permit construction of a detailed genetic blueprint that illustrates the developmental events and hierarchy of genes that govern growth and development of chickens. This review will briefly describe the recent acquisition of chicken genomic resources (ESTs and microarrays) and our consortium's efforts to help launch the new era of functional genomics in the chicken. Copyright © 2004 John Wiley & Sons, Ltd.

In mammals, thyroid hormone responsive Spot 14 (THRSP) is a small acidic protein that is predominately expressed in lipogenic tissue (i.e., liver, abdominal fat and the mammary gland). This gene has been postulated to play a role in lipogenesis, since it responds to thyroid hormone stimulation, high glucose levels and it is localized to a chromosomal region implicated in obesity. In this paper, we report the identification and characterization of duplicated polymorphic paralogs of Spot 14 in the chicken, THRSPα and THRSPβ. Despite low similarity in amino acid (aa) sequence between chickens and mammals, other properties of Spot 14 (i.e., pI, subcellular localization, transcriptional control and functional domains) appear to be highly conserved. Furthermore, a synteny group of THRSP and its flanking genes [NADH dehydrogenase (NDUFC2) and glucosyltransferase (ALG8)] appears to be conserved among chickens, humans, mice and rats. Polymorphic alleles, involving a variable number of tandem repeats (VNTR), were discovered in the putative protein coding region of the duplicated chicken THRSPα (9 bp) and THRSPβ (6 or 12 bp) genes. Our study shows that the THRSPα locus is associated with abdominal fat traits in a broiler×Leghorn resource population.

The goal of our current consortium project is to launch a new era--functional genomics of poultry--by providing genomic resources [expressed sequence tags (EST) and DNA microarrays] and by examining global gene expression in target tissues of chickens. DNA microarray analysis has been a fruitful strategy for the identification of functional genes in several model organisms (i.e., human, rodents, fruit fly, etc.). We have constructed and normalized five tissue-specific or multiple-tissue chicken cDNA libraries [liver, fat, breast, and leg muscle/epiphyseal growth plate, pituitary/hypothalamus/pineal, and reproductive tract (oviduct/ovary/testes)] for high-throughput DNA sequencing of EST. DNA sequence clustering was used to build contigs of overlapping sequence and to identify unique, non-redundant EST clones (unigenes), which permitted printing of systems-wide chicken DNA microarrays. One of the most promising genetic resources for gene exploration and functional gene mapping is provided by two sets of experimental lines of broiler-type chickens developed at INRA, France, by divergent selection for extremes in growth traits (fast-growing versus slow-growing; fatness versus leanness at a similar growth rate). We are using DNA microarrays for global gene expression profiling to identify candidate genes and to map growth, metabolic, and regulatory pathways that control important production traits. Candidate genes will be used for functional gene mapping and QTL analysis of F2 progeny from intercrosses made between divergent genetic lines (fat x lean lines; fast-growing x slow-growing lines). Using our first chicken liver microarray, we have already identified several interesting differentially expressed genes in commercial broilers and in divergently selected broiler lines. Many of these candidate genes are involved in the lipogenic pathway and are controlled in part by the thyrotropic axis. Thus, genome-wide transcriptional profiling is a powerful tool used to visualize the cascade of genetic circuits that govern complex biological responses. Global gene expression profiling and QTL scans should enable us to functionally map the genetic pathways that control growth, development, and metabolism of chickens. This emerging technology will have broad applications for poultry breeding programs (i.e., use of molecular markers) and for future production systems (i.e., the health and welfare of birds and the quality of poultry products).

Although excessive adiposity has become a major drawback in meat type chicken production, few of the genes involved in this process have been characterized so far. In order to identify putative genes involved in adiposity, we performed differential display analysis of RNAs extracted from the liver of divergently selected lean and fat chickens. Twenty-six differential products were selected and purified by single strand conformation polymorphism gel electrophoresis before sequencing and Northern blot analyses. An orthologous sequence of a mammalian cytochrome P450 2C subfamily member was proven to be differentially expressed in the liver of lean and fat chickens and could play an important role in the regulation of adiposity. In mammals, these genes are involved in detoxification of xenobiotics and metabolism of some important biological compounds. Four other genes were found differentially expressed to a lower extent. Some unidentified products were shown to be lean or fat specific, with sequence polymorphism and liver specific expression, strongly suggesting that the related gene could be directly involved in adiposity. Our data indicate that differential display can evidence genes with differential expression and with sequence polymorphism, making this strategy more accurate for differential analysis of messenger RNAs.

s of papers 63 265 Calcium mobilization in the aging hen: II. Effect of the anti-estrogen, tamoxifen, on duodenal calcium absorption. K.K. Franzen*1, M.M. Beck1, and L.G. Robeson1, 1University of Nebraska-Lincoln, Lincoln, NE. Numerous studies have been conducted in an attempt to understand the estrogen/calcium relationship and its effect on optimizing egg production and shell quality. Calcium homeostasis in the laying hen is intimately tied to plasma estrogen profiles. Increases in plasma estrogen concentrations, either endogenous or exogenous, leads to increased calcium absorption and plasma Ca2+ (Sommerville et al., 1989; Qin & Klandorf, 1995; Elaroussi et al., 1993; Hansen, 1998). Two studies were conducted using a potent anti-estrogen, tamoxifen, in an effort to further elucidate the estrogen/calcium relationship in the hen and to determine whether estrogen’s effect on calcium metabolism is mediated through its receptor. HyLine W36 laying hens at peak production (PP; 33 wks of age, 93% production) and late stage production (LS; >85 wks of age, 80% production) were used in this study. Tamoxifen was dissolved in propyleneglycol and administered i.m. 2-3 hours after oviposition and again 8 hours later. Hens were palpated for presence of an egg in the shell gland, and a blood sample for E2, P4 and LH determinations was drawn from the brachial vein of all birds 7 hours after the final tamoxifen injection (4-6h prior to expected oviposition). Hens were then euthanized by cervical dislocation and the duodenal loop was excised for in vitro calcium transport (CaT) determination. Tamoxifen treatment had no effect on plasma E2 or LH concentrations in either study. However, tamoxifen treatment resulted in a significant decrease in plasma P4 concentrations in PP hens (P=0.0455). There was a significant (P<0.05) increase in CaT in tamoxifen treated birds as compared to control birds in both studies. This observation suggests two possibilities: First, that tamoxifen may not be a ”pure” antagonist in the chicken and second, that estrogen may act in a rapid, nongenomic fashion at the intestine. In the latter case, this finding would explain earlier findings that E2 implants caused a rapid, transient increase in CaT that occurs more rapidly than would be expected from an effect via the kidney.

S POULTRY SCIENCE ASSOCIATION August 8–11, 2002 Newark, Delaware * Author Presenting Paper Ancillary Scientists Symposium Genetic Technology Applied to Poultry Production 1 Genetic markers and their application in poultry breeding. M. G. Emara*1, H. Kim1, C. J. Schmidt1, K. S. Decker1, and H. S. Lillehoj2, 1University of Delaware, 2USDA-ARS, PBESL. The current chicken genetic map contains at least 1,965 loci within 50 linkage groups and it covers about 4,000 cM. About 235 of these loci have homology with known human or mammalian genes. The remaining loci are anonymous molecular DNA markers, including microsatellites, AFLP, RAPD, CR1 and others. A third generation genetic map for human research uses single nucleotide polymorphisms (SNPs), which has allowed mapping of complex traits by linkage disequilibrium analysis. With the tremendous advancement in characterizing chicken expressed sequences (ESTs), identification of genetic polymorphisms such as SNPs in chicken genes has become a reality. Our laboratories have undertaken an in silico process using a Phred/Phrap/Polyphred/Consed pipeline to identify candidate chicken SNPs in ESTs. Initial scanning of 23,427 chicken ESTs identified 1,219 candidate SNPs, and validation of the SNPs is ongoing. Placement of SNPs on the chicken genetic map will enhance marker density, thus allowing for fine mapping of complex traits. Application of genetic markers, including SNPs to identify disease resistance genes in commerical broilers will be discussed with regards to coccidiosis.

In order to provide information on chicken genome expression, expressed sequence tags (ESTs) were developed from chicken liver RNAs using a method based on arbitrarily primed reverse transcription-polymerase chain reaction (RT-PCR) of total RNAs. The method is similar to differential display, using one base anchored oligo-d(T) reverse-primers and 20-mer arbitrary forward-primers. A purification step by single strand conformation gel electrophoresis was added before sequencing. With a ratio of 112 unique sequences out of 155, we found this method to be highly effective when compared with EST production with randomly selected clones from non-subtracted, non-normalized libraries. A large proportion of the ESTs sequenced correspond to genes involved in transcriptional and post-transcriptional events. Cytogenetic mapping was performed for a subset of ESTs and four regions of conserved synteny between chicken and human were confirmed.