Growth of embryo and gene expression of nutrient transporters in the small intestine of the domestic pigeon (Columba livia)

Chen et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2015 16(6):511-523 511 Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology) ISSN 1673-1581 (Print); ISSN 1862-1783 (Online) www.zju.edu.cn/jzus; www.springerlink.com E-mail: Growth of embryo and gene expression of nutrient transporters in the small intestine of the domestic pigeon (Columba livia)* Ming-xia CHEN§1, Xiang-guang LI§1, Jun-xian YANG1, Chun-qi GAO1, Bin WANG2, Xiu-qi WANG†‡1, Hui-chao YAN†‡1 (1College of Animal Science, South China Agricultural University / Key Laboratory of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture / Guangdong Provincial Key Laboratory of Agro-Animal Genomics / South China Collaborative Innovation Center for Poultry Disease Control and Product Safety, Guangzhou 510642, China) (2Guangdong Fengli Agricultural Comprehensive Development Co., Ltd., Maoming 525000, China) † E-mail: ; Received Nov. 30, 2014; Revision accepted Mar. 18, 2015; Crosschecked May 13, 2015 Abstract: The objective of this study was to investigate the relationship between gene expression of nutrient (amino acid, peptide, sodium and proton) transporters in the small intestine and embryonic growth in domestic pigeons (Columba livia). One hundred and twenty-five fertilized eggs were randomly assigned into five groups and were incubated under optimal conditions (temperature of 38.1 °C and relative humidity of 55%). Twenty embryos/birds from each group were sacrificed by cervical dislocation on embryonic day (E) 9, 11, 13, 15 and day of hatch (DOH). The eggs, embryos (without yolk sac), and organs (head, brain, heart, liver, lungs, kidney, gizzard, small intestine, legs, and thorax) were dissected, cleaned, and weighed. Small intestine samples were collected for RNA isolation. The mRNA abundance of intestinal nutrient transporters was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR). We classified these ten organs into four types according to the changes in relative weight during embryonic development. In addition, the gene expression of nutrient transporters was differentially regulated by em0,+ + bryonic day. The mRNA abundances of b AT, EAAT3, y LAT2, PepT1, LAT4, NHE2, and NHE3 increased linearly with age, whereas mRNA abundances of CAT1, CAT2, LAT1, EAAT2, SNAT1, and SNAT2 were increased to higher levels on E9 or E11 and then decreased to lower levels until DOH. The results of correlation analysis showed that the 0,+ + gene expressions of b AT, EAAT3, PepT1, LAT4, NHE2, NHE3, and y LAT2 had positive correlations with body weight (0.71<correlation coefficient (CC)<0.82, P<0.0001), while CAT1, CAT2, EAAT2, SNAT1, and SNAT2 had 0,+ negative correlations with body weight (−0.86<CC<−0.64, P<0.0001). The gene expressions of b AT, EAAT3, LAT4, + PepT1, NHE2, NHE3, and y LAT2 showed positive correlations with intestinal weight (0.80<CC<0.91, P<0.0001), while CAT1, CAT2, and EAAT2 showed negative correlations with intestinal weight (−0.84<CC<−0.67, P<0.0001). It was concluded that the differences between growth trajectories of organs and gene expression of nutrient transporters in small intestine were due to their functional and physiological properties, which provided a comprehensive study of amino acid and peptide transporter mRNA in the small intestine during embryonic growth of pigeons. Key words: Embryonic growth, Nutrient transporters, Gene expression, Small intestine, Pigeons doi:10.1631/jzus.B1400340 Document code: A CLC number: S836 1 Introduction ‡ Corresponding authors § The two authors contributed equally to this work * Project supported by the Spark Program of Guangdong, China (No. 2012A020603012) ORCID: Xiu-qi WANG, http://orcid.org/0000-0003-2033-9485 © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 Embryonic growth in precocial birds has been widely investigated, for instance duck (Tangara et al., 2010), turkey (Stepińska et al., 2012), and chicken (Li et al., 2014). In contrast to precocial poultry, young pigeons, a typical representative of altricial birds, are 512 Chen et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2015 16(6):511-523 feed with pigeon milk from the crop by parents for a few days. However, there is limited information available on the development of embryo and organs in this species. The embryonic growth and development in poultry not only directly affect the embryonic quality and hatchability, but also affect the ontogeny of the young after hatch. Unlike mammals, avian embryo development is dependent upon the finite nutrient deposits in the fertile egg (Finkler et al., 1998). Embryonic development of chicks is divided into three phases. At the first third of incubation (first week of incubation), the germ, chorionic sac, and allantoic cavity are established. Embryo completion is formed through the second third (from Day 8 to Day14), and the embryo development is prepared for emergence and hatching in the final stage of incubation (from Day 15 to the day of hatch (DOH)) (Moran, 2007). In the last 2 to 3 d of incubation, the residual yolk, as a nutritional source, reaches the small intestine via the yolk stalk (Noy and Sklan, 1998). Therefore, the embryo would impair absorption of nutrients from the yolk sac, and then nutrient absorption occurs primarily through the small intestine close to hatch (Noy et al., 1996; Speier et al., 2012). Thus, it is necessary to understand the development patterns of intestinal absorptive capacity because of its key function in nutrient intake. In the small intestine, the absorption of nutrients is mediated by transporter proteins expressed in the enterocyte. The nutrient passed through the epithelium of the small intestine and into the blood stream via special transporters. According to Poncet and Taylor (2013), different nutrient transporters have different gene expression patterns and functions. For example, amino acids are transported into the intestinal epithelial cells as di- or tri-peptides by the H+-dependent peptide transporter 1, PepT1 (Leibach and Ganapathy, 1996) or a variety of amino acid transporters that have specificity for anionic, cationic, or neutral amino acids (Hyde et al., 2003). Most of the studies are focused on the gene expressions of intestine nutrient transporters in precocial poultry, such as chicken (Mott et al., 2008; Sun et al., 2015; Miska et al., 2014) and turkey (de Oliveira et al., 2009). Our laboratory has studied gene expressions of cationic and neutral amino acid (Zeng et al., 2011), excitatory amino acid (Li et al., 2011), small peptide, and sodium and proton (Li et al., 2012) transporters in the small intestine of chick embryos. Furthermore, these studies found that the expression levels of the b0,+AT, EAAT3, and PepT1 in the small intestine had strong correlations with chick embryo development, which could be used as indicators for the chick embryo growth of the small intestine (Li et al., 2013). The gene (...truncated)