MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY Prehatch intestinal maturation of turkey embryos demonstrated through gene expression patterns J. E. de Oliveira ,*1 S. Druyan ,*2 Z. Uni ,† C. M. Ashwell ,* and P. R. Ferket *3 * North Carolina State University, Department of Poultry Science, Raleigh 27695; and † Hebrew University, Faculty of Agriculture, Department of Animal Science, Rehovot, Israel 76100 ABSTRACT Some of the challenges faced by neonatal by gene expression pattern similarity into 4 groups. turkeys include weakness, reduced feed intake, impaired The expression pattern of hormone receptors revealed growth, susceptibility to disease, and mortality. These that intestinal tissues may be less responsive to growth symptoms may be due to depleted energy reserves after hormone, insulin, glucagon, and triiodothyronine dur- hatch and an immature digestive system unable to re- ing the last 48 h before hatch, when developmental plenish energy reserves from consumed feed. To better emphasis switches from cell proliferation to functional understand enteric development in turkeys just before maturation. Based on gene expression patterns, we con- hatch, a new method was used to identify the patterns cluded that at hatch, poults should have the capacity of intestinal gene expression by utilizing a focused mi- to 1) digest disaccharides but not oligopeptides, due croarray. The duodenums of 24 turkey embryos were to increased expression of sucrase-isomaltase but de- sampled on embryonic day (E)20, E24, E26, and hatch creased expression of aminopeptidases and 2) absorb (E28). The RNA populations of 96 chosen genes were monosaccharides and small peptides due to high ex- measured at each time point, from which 81 signifi- pression of sodium-glucose cotransporter-4 and peptide cantly changed (P < 0.01). These genes were clustered transporter-1. Key words: turkey embryo , intestinal development , gene expression , microarray 2009 Poultry Science 88 :2600–2609 doi: 10.3382/ps.2008-00548 INTRODUCTION In order for poults to independently forage and digest food immediately after hatch, they must develop before Modern turkey strains have been selected for fast hatch the necessary complement of digestive enzymes growth, efficient feed conversion, and high meat yield and mucosal maturity with the absorptive capacity to (Havenstein et al., 2007). Moreover, the time to raise use ingested feed. This prenatal development of diges- a turkey to market size decreases each year, and thus tive capacity increases as the avian embryos orally neonatal growth and development become an increasing consume their amniotic fluid before hatch (Romanoff, proportion of the productive life of a turkey (Uni and 1960, 1967; Uni and Ferket, 2004; Moran, 2007). The Ferket, 2004). Some of the challenges neonatal turkeys nutrients within the amnion comprise the first meal must overcome include weakness due to low feed intake of the embryo and along with yolk infusion into the or feed refusal (Christensen et al., 2003). Poor digestion intestine (Esteban et al., 1991a,b; Noy et al., 1996), of feed and malabsorption increases the susceptibility they facilitate enteric development toward hatch (Uni of poults to opportunistic pathogens, resulting in im- et al., 2003), but the details of this development are not paired growth and high “starve out” mortality (Barnes, clearly understood. 1994a,b). During the first few days after hatch, turkey The perinatal development of digestive capacity can be poults often succumb to low energy status (Donaldson, evaluated by studying the activity and gene expression 1995), most likely due to an immature digestive sys- of digestive enzymes and nutrient transporters in the tem (Uni and Ferket, 2004; Christensen et al., 2007). intestine. Pancreatic enzymes carboxypeptidase A and chymotrypsin have been reported in chicken embryos at © 2009 Poultry Science Association Inc. 16 d of incubation or embryonic day (E)16 (Marchaim Received December 15, 2008. and Kulka, 1967). Activities of maltase, aminopepti- Accepted September 3, 2009. 1 Current address: Provimi Research and Innovation Centre, Len- dase (XPNPEP1), sodium-glucose cotransporter-1 neke Marelaan 2, Sint-Stevens-Woluwe, B1932, Belgium. (SGLT1), and ATPase ion channel (P2X1) were found 2 Current address: Institute of Animal Science, Agricultural Re- in chicken embryos on E19, and their mRNA expression search Organization, The Volcani Center, PO Box 6, Bet Dagan 50250, Israel. was detected as early as E15 (Uni et al., 2003). Activity 3 Corresponding author: [email protected] of the brush border enzymes leucine amino peptidase 2600 TURKEY EMBRYO INTESTINAL GENE EXPRESSION 2601 and sucrase-isomaltase, as well as jejunum glucose and North Carolina State University Institutional Animal alanine uptake, have been reported in turkey embryos Care and Use Committee. as early as E25 (Foye et al., 2007). Extensive studies on chicken intestinal enzymes and nutrient transporters Statistical Analyses from E18 to 14 d posthatch have been recently pub- lished (Gilbert et al., 2007; Frazier et al., 2008; Mott et In turkey embryos, differences between day of incu- al., 2008). However, there is limited knowledge about bation in YFBW, DYFBW, and duodenum weight data at which phases of embryonic development these genes were tested by 1-way ANOVA according to the follow- start to be expressed, especially in turkeys. A better ing model: Y = μ + Age + e [equation 1], where Y understanding of small intestine maturation and gene = weight; μ = mean; and e = random error, with the expression pattern before hatch may help turkey nutri- fixed effect of age (E20, E22, E24, E26, or E28). All of tionists formulate diets that complement the digestive the statistical analyses were conducted using the JMP capacity at hatch. Genes of interest must include diges- software (SAS Institute, 2005). tive brush border enzymes for carbohydrate, protein and lipid, nutrient transporters, and hormone receptors RNA Isolation, Sample Labeling, involved in intestinal tissue growth, maturation, and and Microarray Procedure function. High-throughput DNA microarrays are able to identify many RNA populations in one single experi- Total RNA was extracted from 100 mg of duodenum ment (Harris, 2000; Spielbauer and Stahl, 2005), quali- tissue using TRI Reagent (Molecular Research Center, fying it as a good tool for the task of identifying the Cincinnati, OH). Equal amounts of RNA from 6 em- pattern of expression of these genes. Even though turkey bryos were pooled and adjusted to 0.5 μg/μL of concen- gene sequences are still not available for much of the tration. Complementary DNA was produced from each genome, it has been demonstrated that gene sequences RNA pool and indirect-labeled either with Cy3 or Cy5 in chickens and turkeys are similar enough that chicken utilizing the ChipShot Indirect Labeling and Clean-up probes can hybridize to turkey target RNA (Reed et System kit (Promega, Madison, WI), according to the al., 2005). More recent data collected for comparative experimental design (Figure 1). Thirty-three picomoles genome organization studies to characterize the physi- of cDNA labeled with each Cy-Dye (Amersham Biosci- cal map for the turkey (Romanov and Dodgson, 2006) ences Corp., Piscataway, NJ) was hybridized in each ar- and the distribution of copy number variants across ray slide for 16 h at 42°C, then was washed and scanned species (chicken:turkey; Griffin et al., 2008) show the to generate digitized image data files. Microarray slides utility of these cross-species hybridizations for studying were produced by the North Carolina State University the turkey genome. The objective of this study was to Domestic Animals Genomic Laboratory. The microar- use microarray technology to map small intestine gene ray platform used was NCSU_Chicken_JS1, registered expression in turkey embryos during the last phase of at the National Center for Biotechnology Informa- incubation from 20 d of incubation until day of hatch. tion Gene Expression Omnibus (GEO) (http://ncbi. nlm.nih.gov/projects/geo) under accession number GPL6041. This is an earlier version of the microarray MATERIALS AND METHODS platform introduced by Druyan et al. (2008), contain- Egg Incubation and Tissue Sampling ing 96 probes. The most important feature of this array is the high number of replication because each gene se- Two hundred Nicholas 300 turkey eggs were ob- quence was spotted 8 times in each slide, and combined tained from a commercial turkey operation (Prestage biological and technical replicates totaled 32 observa- Farms, Clinton, NC) and were incubated under stan- tions per gene. An additional feature of these studies is dard conditions. At E19, eggs were candled, infertile the cross-species hybridization of turkey cDNA with the eggs and dead embryos were removed, and the remain- chicken sequence-based arrays. To validate the utility of ing eggs were divided into 5 groups of 24 eggs with this platform for turkey studies, we performed a bioin- similar weight distribution (74.3 ± 10 g). One group of formatic analysis (BLASTN via http://blast.ncbi.nlm. 24 eggs was sampled at E20, E22, E24, E26, and E28 nih.gov/Blast.cgi) of the chicken 70-mer oligos printed (hatch). The eggs were opened at the blunt end using on the arrays to the available gene sequences for tur- surgical scissors; the embryo was extracted and killed key. In addition, a comparative hybridization of pooled by cervical dislocation. Embryos were weighed with- RNA from liver and intestine (1:1) was