家鸽胚胎的生长与其小肠营养素转运载体基因表达

Growth of embryo and gene expression of nutrient transporters in the small intestine of the domestic pigeon ( Columba livia ) 家鸽胚胎的生长与其小肠营养素转运载体基因表达 1 first-author 1 1 1 2 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:xqwang@scau.edu.cnyanhc@scau.edu.cn †E-mail:xqwang@scau.edu.cnyanhc@scau.edu.cn xqwang@scau.edu.cn 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:xqwang@scau.edu.cnyanhc@scau.edu.cn †E-mail:xqwang@scau.edu.cnyanhc@scau.edu.cn xqwang@scau.edu.cn College 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 College 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 Guangdong Fengli Agricultural Comprehensive Development Co., Ltd., Maoming 525000, China Guangdong Fengli Agricultural Comprehensive Development Co., Ltd., Maoming 525000, China 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 embryonic day. The mRNA abundances of b 0,+ 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 gene expressions of b 0,+ 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 negative correlations with body weight (−0.86 < CC < −0.64, P < 0.0001). The gene expressions of b 0,+ 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. 目的 2 探索家鸽胚胎期器官生长发育及其小肠营养素转运载体基因表达规律，为家鸽胚胎期的营养调控提供理论基础。 创新点 2 第一次较全面地研究了家鸽胚胎期小肠氨基酸转运载体基因表达规律。 方法 2 利用实时荧光定量逆转录聚合酶链反应（real-timeRT-PCR）技术，同时结合了多内标校正方法对小肠营养素转运载体基因表达模式进行分析。 结论 2 在家鸽胚胎期发育的过程中，不同的器官生长模式不完全一致。营养素转运载体 b0,+ AT 、 EAAT3 、 LAT4 、 PepT1 、 NHE2 、 NHE3 和 y + LAT2 基因的表达与小肠重量呈正相关关系， CAT1 、 CAT2 和 EAAT2 基因的表达与小肠重量呈负相关关系。 Embryonic growth Nutrient transporters Gene expression Small intestine Pigeons 胚胎生长 营养素转运载体 基因表达 小肠 家鸽 Introduction 1 Embryonic growth in precocial birds has been widely investigated, for instance duck (Tangara et al., 2010 2010 ), turkey (Stepińska et al., 2012 2012 ), and chicken (Li et al., 2014 2014 ). In contrast to precocial poultry, young pigeons, a typical representative of altricial birds, are 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 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 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 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 1996 ; Speier et al., 2012 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 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 1996 ) or a variety of amino acid transporters that have specificity for anionic, cationic, or neutral amino acids (Hyde et al., 2003 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 2008 ; Sun et al., 2015 2015 ; Miska et al., 2014 2014 ) and turkey (de Oliveira et al., 2009 2009 ). Our laboratory has studied gene expressions of cationic and neutral amino acid (Zeng et al., 2011 2011 ), excitatory amino acid (Li et al., 2011 2011 ), small peptide, and sodium and proton (Li et al., 2012 2012 ) transporters in the small intestine of chick embryos. Furthermore, these studies found that the expression levels of the b 0,+ 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 2013 ). The gene expression level of intestinal fatty acid (Xie et al., 2012 2012 ; 2013 2013 ) and sodium glucose (Dong et al., 2012b 2012b ) transporters in pigeons, and the activity of digestive enzyme in pigeon yolk sac membrane during pre- and post-hatch development have been reported (Dong et al., 2012a 2012a ). However, the underlying relationship of embryonic growth and gene expression of nutrient transporters in the small intestine has not been evaluated in pigeons. Therefore, the objective of this study was to investigate: (1) the growth of embryo and organs in the stage of embryonic development and (2) the underlying relationship between gene expression of nutrient (amino acid, peptide, sodium and proton) transporters in the small intestine and embryonic growth of pigeons. Materials and methods 1 All experimental methods and management procedures were approved by the Animal Care Committee of South China Agricultural University (Guangzhou, China). Experimental design and sample collection 2 A total of 125 fertilized pigeon ( Columba livia ) eggs were obtained from Brother Economic Development Co. Ltd. (Guangdong, China). Eggs were weighed ((21.08±0.12) g) and randomly assigned into five groups. The egg weights in each group did not show a significant difference ( P > 0.90). The eggs were then incubated at 38.1 °C and 55% relative humidity in an incubator (Keyu, Guangzhou, China). At embryonic day (E) 7 of incubation, all the eggs were candled for the detection of unfertilized eggs; these were removed from the incubator. On E9, E11, E13, E15, and DOH, the eggs were opened and embryos/birds were freed from the allantois and yolk. At any given age, twenty embryos/birds from each group were sacrificed by cervical dislocation. The eggs, embryos (without yolk sac), and organs (head, brain, heart, liver, lung, kidney, gizzard, small intestine, leg, and thorax) were dissected, lightly blotted and drained of blood or any fluid contents with phosphate-buffered saline (NaCl, 8 g/L; KCl, 0.2 g/L; Na 2 HPO 4 , 1.42 g/L; KH 2 PO 4 , 0.27 g/L) at 4 °C, and weighed. All of the small intestine samples were placed in 0.5-ml microcentrifuge tubes, frozen in liquid nitrogen, and stored at −80 °C for mRNA analysis. Total RNA isolation and cDNA synthesis 2 The total RNA was isolated from the small intestine tissue of pigeon embryos using TRIzol Reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. The RNA quality was checked by 1.5% agarose gel electrophoresis, stained with 10 μg/ml ethidium bromide. The optical density (OD) of the RNA was measured by a NanoDrop 2000 UV-Vis spectrophotometer (Thermo, Wilmington, MA, USA) and OD 260 /OD 280 ratio was between 1.8 and 2.0. Reverse transcription (RT) reaction mixtures (20 μl) comprised 2 μg of total RNA, N10 primers (Sangon, Shanghai, China), M-MLV reverse transcriptase (Promega, Madison, USA), RNase inhibitor (TaKaRa, Shiga, Japan), and dNTP. The complementary DNA (cDNA) products were diluted 1:3 using DEPC water and stored at 20 °C for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Quantification of mRNA by qRT-PCR 2 The abundance of mRNA was measured by qRT-PCR with a Stratagene MxPro 3005P thermocycler (Agilent Technologies, Santa Clara, USA). The qRT-PCR mixture contained 2 μl of cDNA template, 10 μl of SYBR Green Real-Time PCR Master Mix (TOYOBO, Tokyo, Japan), 7 μl of DEPC water, and 1 μl of each gene-specific primer. The PCR program was 1 cycle at 95 °C for 60 s, 35 cycles of 95 °C for 15 s, 58 °C/63 °C for 40 s, and 72 °C for 40 s, 95 °C for 60 s, 58 °C/63 °C for 15 s, and 72 °C for 30 s. The melting curve analysis was performed by 1 cycle at 95 °C for 60 s, 58 °C/63 °C for 15 s, and 72 °C for 30 s, starting at 58 °C with a heating rate of 0.2 °C per second up to 95 °C. The cycle threshold ( C T ) values for each gene were averaged. The following nutrient transporters were investigated in our study: solute carrier (SLC) family 1 member 2 ( EAAT2 ), SLC family 1 member 1 ( EAAT3 ), SLC family 7 member 1 ( CAT1 ), SLC family 7 member 2 ( CAT2 ), SLC family 7 member 6 ( y + LAT2 ), SLC family 7 member 9 ( b 0,+ AT ), SLC family 7 member 5 ( LAT1 ), SLC family 43 member 2 ( LAT4 ), SLC family 38 member 1 ( SNAT1 ), SLC family 38 member 2 ( SNAT2 ), SLC family 15 member 1 ( PepT1 ), SLC family 9 member 2 ( NHE2 ), and SLC family 9 member 3 ( NHE3 ). Six housekeeping genes, α-tubulin , glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), β-actin , TATA box binding protein ( TBP ), ribosomal protein L13 ( RPL13 ), and hypoxanthine phosphoribosyltransferase 1 ( HPRT1 ) were also evaluated. All the gene-specific primers were designed by Primer Premier 5.0 software (Premier Biosoft, Palo Alto, CA, USA). Table 1 1 shows the primer sequences used. b 0,+ AT : solute carrier (SLC) family 7 member 9; CAT1 : SLC family 7 member 1; CAT2 : SLC family 7 member 2; EAAT2 : SLC family 1 member 2; EAAT3 : SLC family 1 member 1; LAT1 : SLC family 7 member 5; LAT4 : SLC family 43 member 2; NHE2 : SLC family 9 member 2; NHE3 : SLC family 9 member 3; PepT1 : SLC family 15 member 1; SNAT1 : SLC family 38 member 1; SNAT2 : SLC family 38 member 2; y + LAT2 : SLC family 7 member 6; GAPDH : glyceraldehyde 3-phosphate dehydrogenase; HPRT1 : hypoxanthine phosphoribosyltransferase 1; RPL13 : ribosomal protein L13; TBP : TATA box binding protein Gene GenBank ID Sense (S)/antisense (AS) primer (5'→3') PCR product length (bp) 4 Transporter gene   b 0,+ AT XM_005514428.1 S: AATCATTGGCTCAGGTATCTT 237   AS: TTTGTGACGAGTAGGCTTG   CAT1 XM_005501421 S: CTTCGCCGTGGTGATA 108   AS: CCGAGGACGAGGATGT   CAT2 XM_005501186 S: TCAAGCGTAGCAAGAGC 232   AS: CGAAGATTAGGACCAAGAT   EAAT2 NM_001282830.1 S: GCCTAAACAAGTAGAAGTGC 140   AS: CCAGGATGACACCGAATA   EAAT3 XM_005507165.1 S: ACAGGTGTTGCTGCTTTG 171   AS: GGTGCTGCCCACTCTAT   LAT1 XM_005504728 S: GCAAAGGGTGATGTGACGAG 170   AS: AGAGGCAGGTTTCTGTAGGGA   LAT4 XM_005502006.1 S: GACCTTCACATCGCTTACG 163   AS: TGCCCAGACAAGCAGAAT   NHE2 XM_005504658 S: ATTGAGCCACTCTTTGTCTT 236   AS: TGGTTCTTCCCAACTGTAG   NHE3 XM_418895.2 S: ATTGCCTCTTTTACCCTTAC 215   AS: CTGTCCAGGTGGCCCATTA   PepT1 XM_005504628.1 S: CTATTCAGATTCAGCCAGACC 250   AS: ATTACTTTGATTTGGGATTGC   SNAT1 XM_005499915 S: TGGGCGGTTTCTTGTTGT 277   AS: GATGGTGGGCAAAGCGTA   SNAT2 XM_005499916 S: GTGGCACCTTTGGTAGATG 129   AS: AATCGCAGGATGGCAGAC   y + LAT2 XM_001231336 S: GCTGAACTTGGAACCACTATTAC 102 AS: AAGTGAGGTCCACAAACGAA 4 Housekeeping gene   α-tublin NM_001080860 S: GAGAACACGGATGAAACCTACT 118   AS: TGGTGGCTGACACTAAATGG   β-actin NM_205518.11 S: ATTGTCCACCGCAAATGCTTC 113   AS: AAATAAAGCCATGCCAATCTCGTC   GAPDH NM_001282835.1 S: TCTGCCAAAGTCCAAGTG 187   AS: ACTTTATGTTGCTGGGAT   HPRT1 XM_005500563.1 S: TACGACCTGGACTTGTTCT 172   AS: ATTTATAGCCACCCTTGAG   RPL13 XM_005504844.1 S: TGTCCAACCATCCGATACCA 263   AS: GTCGCCATCTTCAGTTCCTCA   TBP XM_005508625.1 S: CAGCCCATTTACCAGG 142 AS: CAACCAAGATTCACCGT Statistical analysis 2 The relative weight of embryo was absolute embryo weight to set egg weight, and the relative weight of organs (head, brain, heart, liver, gizzard, small intestine, kidney, lung, leg, and thorax) was measured by the absolute organ weight to set yolk-free embryo weight. The obtained C T values of housekeeping genes were quantified and entered into the geNorm application according to the method described by Vandesompele et al. ( 2002 2002 ). The average expression stability (M) values of control genes calculated by geNorm showed that GAPDH , and β-actin , TBP , and HPRT1 were the most stable genes among GAPDH , β-actin , HPRT1 , TBP , RPL13 , and α-tubulin . In addition, the pair-wise variation value V 4/5 was below 0.15 and the normalization factors utilized GAPDH , β-actin , TBP , and HPRT1 as housekeeping genes. The relative mRNA expression levels were calculated by the method of 2 −Δ C T and revised through normalization factors. Data were processed by SAS version 9.1.3 statistical software package (SAS Institute Inc., Cary, NC, USA). The correlation coefficients (CCs) were derived from the Pearson product-moment correlation using a CORR Procedure analysis. The regression analysis was performed by the REG Procedure. A linear model, power function or logarithmic function was decided according to the scatter diagram plot, which used a least-square regression analysis method of the REG procedure by a stepwise variable filter method. Values were expressed as the mean±standard error (SE). Differences with P < 0.05 and P < 0.01 were considered statistically significant and highly significant, respectively. Results 1 Growth of embryo and organs 2 The absolute weight of yolk-free embryo increased from 1.27 g on E9 to 11.47 g on DOH (Fig. 1 1 ). The relative embryo weight increased significantly throughout development, with a sharp increase from E13 to E15. According to the changes of the relative weight, ten organs were classified into four groups during the embryonic developmental stage. The head and brain were classified as the first type of organs with a gradually decreasing relative weight (Fig. 2 2 ), whereas the kidney, gizzard, and leg were classified as the second type of organ with a continuously rising relative weight (Fig. 3 3 ). The heart, small intestine, and liver were classified as the third type of organ with generally increasing relative weight from E9 to DOH but a more rapid increase near the end of incubation (Fig. 4 4 ). The lung and thorax were classified as the fourth type of organ with initially increasing relative weight, peaking on E11 and E13, respectively, and then decreasing (Fig. 5 5 ). Relative weight is calculated as absolute weight normalized to fresh egg weight (%). Values are expressed as mean±SE ( n =20). Data with no common letters (a–e) indicate significant difference among embryonic days ( P< 0.05) Relative organ weight is calculated as absolute weight normalized to embryonic body weight without yolk (%). Values are expressed as mean±SE. Data with no common letters (a–e) indicate significant difference among embryonic days ( P < 0.05) Relative organ weight is calculated as absolute weight normalized to embryonic body weight without yolk (%). Values are expressed as mean±SE. Data with no common letters (a–e) indicate significant difference among embryonic days ( P < 0.05) Relative organ weight is calculated as absolute weight normalized to embryonic body weight without yolk (%). Values are expressed as mean±SE. Data with no common letters (a–e) indicate significant difference among embryonic days ( P < 0.05) Relative organ weight is calculated as absolute weight normalized to embryonic body weight without yolk (%). Values are shown as mean±SE. Data with no common letters (a–e) indicate significant difference among embryonic days ( P < 0.05) Correlation of organ and body weight 2 Correlation analysis of organ and body weight is shown in Table 2 2 . During embryonic development, the weights of head, brain, heart, liver, lung, kidney, gizzard, small intestine, thorax, and legs were strongly positively correlated with body weight (CC > 0.8, P < 0.0001). Yolk was negatively correlated with body weight (CC=−0.637, P < 0.0001). In addition, we performed regression analysis on organ and body weight (Table 2 2 ). A linear model, power function or logarithmic function with organ weight was represented as a function of embryonic body weight according to the scatter diagram plot ( n =112), which used a least-square regression analysis method of the REG Procedure by a stepwise variable filter method 2 Weight of organs ( Y ) 2 Embryonic body weight of pigeon ( X , g) Correlation coefficient ( P -value) Regression model ( R 2 )  Head 0.973 (<0.0001) Y =0.54 X 0.61 (0.9874)  Brain 0.987 (<0.0001) Y =54.71×10 −3 X 0.76 (0.9819)  Yolk −0.637 (<0.0001) Y =1.14ln X (0.8164)  Thorax 0.968 (<0.0001) Y =0.19ln X (0.9861)  Leg 0.971 (<0.0001) Y =16.41×10 −3 X (0.9815)  Heart 0.946 (<0.0001) Y =6.27×10 −3 X 1.13 (0.9544)  Liver 0.903 (<0.0001) Y =15.30×10 −3 ×1.33 X (0.9661)  Lung 0.925 (<0.0001) Y =0.07ln X (0.9860)  Kidney 0.952 (<0.0001) Y =3.02×10 −3 X 1.66 (0.9731)  Gizzard 0.981 (<0.0001) Y =12.26×10 −3 X 1.47 (0.9920)  Small intestine 0.894 (<0.0001) Y =4.01×10 −3 X 1.74 (0.9655) A linear model, power function or logarithmic function with organ weight was represented as a function of embryo weight according to the scatter diagram plot. The results indicated that organs have their special development patterns. Correlation of embryo weight and embryonic day 2 On Day 16 of incubation, eggs were checked every 3 h to determine time of external pipping and hatching, and the DOH was on E17.5. The correlation of embryo weight and embryonic day was measured. The result showed that the body weights had linearly correlated with the embryonic day (CC=0.978, P < 0.0001) and the equation of linear regression was Y =−10.59+1.26 X ( R 2 =0.9606; Y was embryo weight of pigeon, and X was embryonic day). Gene expression of nutrient transporters in small intestine 2 In our study, gene expressions for ten amino acid transporters ( EAAT3 , EAAT2 , b 0,+ AT , y + LAT2 , CAT1 , CAT2 , LAT1 , LAT4 , SNAT1 , and SNAT2 ), two sodium and proton transporters ( NHE2 and NHE3 ), and a peptide transporter ( PepT1 ) were measured. Compared with the housekeeping genes, GAPDH , β-actin , TBP , and HPRT1 , the nutrient transporter expression patterns were analyzed on E9, E11, E13, E15, and DOH in the small intestine of embryonic pigeons. The results indicated that genes of EAAT3 , b 0,+ AT , y + LAT2 , PepT1 , LAT4 , NHE2 , and NHE3 showed similar expression profiles in the small intestine with a gradual increase in the intestine from E9 to DOH and a rapid rise during late embryogenesis (Table 3 3 ). The gene expressions of CAT1 , CAT2 , LAT1 , EAAT2 , SNAT1 , and SNAT2 exhibited similar profiles in the small intestine, with higher levels on E9 or E11, which then decreased to lower levels until the end of incubation (Table 3 3 ). E: embryonic day. GAPDH , β-actin , TBP and HPRT1 are used for normalization as housekeeping genes. Data are shown as mean±SE ( n =12). Different letters (a–e) within the same row indicate significant differences among embryonic day ( P < 0.05) 2 Gene 5 Gene expression (×10 −3 ) E9 E11 E13 E15 DOH   b 0,+ AT 2.76±0.26 b 9.12±1.71 b 8.75±0.79 b 58.43±8.29 b 603.21±52.28 a   CAT1 358.37±18.00 b 460.62±30.62 a 350.16±22.65 b 163.62±12.66 c 53.24±5.50 d   CAT2 449.87±45.35 b 651.16±71.66 a 362.22±22.33 b 151.00±14.48 c 72.42±5.63 c   EAAT2 403.76±48.53 a 429.48±38.33 a 374.95±32.88 a 136.05±7.77 b 67.81±4.64 b   EAAT3 35.95±4.83 d 65.77±4.91 d 135.10±18.10 c 222.63±14.53 b 363.88±40.61 a   LAT1 353.45±42.27 a 287.8±21.51 ab 246.56±29.82 b 148.05±17.3 c 72.71±5.44 c   LAT4 52.58±2.44 d 72.76±5.15 cd 99.97±5.06 bc 121.78±13.06 b 597.92±30.59 a   NHE2 37.25±4.58 b 55.68±7.13 b 28.09±3.22 b 64.48±10.43 b 703.42±46.41 a   NHE3 22.40±3.02 b 33.43±8.71 b 25.55±2.75 b 78.89±8.66 b 420.75±58.39 a   PepT1 4.96±0.70 c 4.42±0.60 c 16.96±1.22 c 88.12±6.58 b 385.96±33.79 a   SNAT1 288.51±23.14 a 54.86±9.88 b 59.22±4.02 b 40.96±2.44 b 28.79±4.09 b   SNAT2 851.41±56.95 a 286.70±51.87 b 185.37±6.49 c 172.85±12.02 c 153.12±8.22 c   y + LAT2 82.92±6.79 c 74.02±4.04 c 88.30±3.26 c 152.20±13.87 b 624.61±30.72 a Correlation of gene expression and weight of body and small intestine 2 The results indicated that the gene expression of EAAT3 and PepT1 had a strong positive correlation with body weight (CC > 0.8, P < 0.0001), but CAT1 had a strong negative correlation with body weight and it had a CC of −0.860. The gene expressions of b 0,+ AT , LAT4 , NHE2 , NHE3 , and y + LAT2 showed positive correlations with body weight (0.6 < CC < 0.8, P < 0.0001), whereas CAT2 , EAAT2 , SNAT1 , and SNAT2 had negative correlations with body weight (−0.8 < CC < −0.6, P < 0.0001). Although the LAT1 gene expression in the embryonic small intestine was not at a low level during the embryonic developmental stage, it had a low correlation with body weight (CC=−0.381, P =0.0035) (Table 4 4 ). A linear model, power function or logarithmic function with embryo weight was represented as a function of gene expression according to the scatter diagram plot ( n =60), which used a least-square regression analysis method of the REG Procedure by a stepwise variable filter method 2 Gene expression ( X ) 2 Embryo weight ( Y ) Correlation coefficient ( P -value) Regression model ( R 2 )   b 0,+ AT 0.743 (<0.0001) Y =1.80ln X +12.80 (0.9021)   CAT1 −0.860 (<0.0001) Y =−3.77ln X +0.46 (0.8009)   CAT2 −0.745 (<0.0001) Y =−3.68ln X +1.16 (0.8346)   EAAT2 −0.790 (<0.0001) Y =−4.14ln X −0.14 (0.8372)   EAAT3 0.820 (<0.0001) Y =24.43 X 0.78 (0.8488)   LAT1 −0.381 (0.0035)   LAT4 0.791 (<0.0001) Y =3.91ln X +13.77 (0.8854)   PepT1 0.821 (<0.0001) Y =1.98ln X +13.35 (0.9585)   NHE2 0.736 (<0.0001) Y =2.47ln X +12.17 (0.7050)   NHE3 0.711 (<0.0001) Y =2.57ln X +14.06 (0.8279)   SNAT1 −0.636 (<0.0001) Y =9.30×(5.59×10 −3 ) X (0.8279)   SNAT2 −0.656 (<0.0001) Y =1.15 X −1.06 (0.8341)   y + LAT2 0.782 (<0.0001) Y =3.75ln X +13.49 (0.7702) The gene expressions of b 0,+ AT , EAAT3 , LAT4 , PepT1 , NHE2 , NHE3 , and y + LAT2 showed positive correlations with intestinal weight (CC > 0.8, P < 0.0001), whereas CAT1 , CAT2 , and EAAT2 showed negative correlations, with CC ranging from −0.841 to −0.672 (Table 5 5 ). However, there was no significant correlation between the gene expression of other genes ( LAT1 , SNAT1 , and SNAT2 ) and intestinal weight. Based on regression analysis, it was shown that different nutrient transporters had different gene expression patterns. This might be attributed to the demand of physiological function. A linear model, power function or logarithmic function with small intestinal weight was represented as a function of gene expression according to the scatter diagram plot ( n =60), which used a least-square regression analysis method of the REG Procedure by a stepwise variable filter method 2 Gene expression ( X ) 2 Small intestinal weight ( Y ) Correlation coefficient ( P -value) Regression model ( R 2 )   b 0,+ AT 0.907 (<0.0001) Y =0.07ln X +0.39 (0.8972)   CAT1 −0.841 (<0.0001) Y =−0.16ln X −0.12 (0.8952)   CAT2 −0.672 (<0.0001) Y =−0.15ln X −0.08 (0.7738)   EAAT2 −0.748 (<0.0001) Y =−0.17ln X −0.12 (0.8167)   EAAT3 0.801 (<0.0001) Y =1.10 X 1.34 (0.8559)   LAT1 −0.104 (0.4408)   LAT4 0.943 (<0.0001) Y =0.18ln X +0.49 (0.9378)   PepT1 0.946 (<0.0001) Y =0.92 X +0.03 (0.9378)   NHE2 0.914 (<0.0001) Y =0.11ln X +0.43 (0.9056)   NHE3 0.864 (<0.0001) Y =0.11ln X +0.45 (0.8845)   SNAT1 −0.455 (0.0004)   SNAT2 −0.469 (0.0003)   y + LAT2 0.942 (<0.0001) Y =0.75 X +0.01 (0.9471) Discussion 1 Development of embryos 2 It was well known that avian embryo growth is affected by breeder age, egg weight, and incubation temperature (Tona et al., 2004 2004 ; Ipek et al., 2014 2014 ). With larger fresh eggs, there are more resources available within the egg’s closed environment for the embryo to use during development. Thus, larger eggs have larger embryos (Al-Murrani, 1978 1978 ). During embryonic development, the absolute weight of pigeon embryo increases constantly with age. The average embryo mass of pigeon on DOH was only (13.36±0.17) g. Compared with precocial poultry, pigeons, as an altricial poultry, had small embryos for the lower energy. One report has stated that growth of embryos varies in direct proportion to egg mass during incubation (Filippi et al., 2012 2012 ). However, to elucidate the changes in embryo, the relative embryo weight was measured during embryonic development. The relative embryo weight increased significantly with age, and the embryo growth in relative terms was increasing approximately up to 60% from E9 to DOH. In addition, to measure the correlation of embryonic day and embryo weight, we presented the data of embryo weight as a function of embryonic day. The result showed that the growth of embryos was linearly correlated with the embryonic day, which indicated that the development of embryos was affected by embryonic day. Development of organs 2 Different organs have different growth trajectories (Figs. 2 2 – 5 5 ). However, some organs have similar growth patterns when normalized by body weight. The head and brain, when normalized by body weight, showed a similar pattern as the first kind of organ with a gradual decrease during embryonic development (Fig. 2 2 ). It is well known that the head and the brain, among the ten organs we measured, are a section of the body renowned for dropping their relative weight during development in many species (Azzam and Mortola, 2007 2007 ). The increased absolute weights of those two organs but their decreased relative weights may be accounted for by their priority development. Li et al. ( 2013 2013 ) and Azzam et al. ( 2007 2007 ) observed similar growth trajectories in the head during chick embryonic development. In contrast to the head and brain, the kidney, gizzard, and leg were classified as the second kind of organ because their relative weight had similar growth patterns, initially low and increasing gradually during embryonic development. The relative weight of kidney was similar to our previous study on chicken, while the gizzard, as one of the digestive organs, showed different development trajectories as described by Li et al. ( 2013 2013 ). In this study, the heart, liver, and small intestine were included as the third kind of organ for the similar growth pattern with a slight increase from E9 to E15 but a rapid rise from E15 to DOH in terms of their relative weights. During embryonic development, nutrition from yolk, digestive tract or blood was stored, absorbed and transformed in the liver and then passed into other organs to utilize. However, in the final stage of incubation, β-oxidation of fatty acids cannot provide the energy needed for embryonic development (Tazawa et al., 1983 1983 ; Moran, 2007 2007 ). Therefore, the development of embryos was mainly driven by glucose metabolism, such as gluconeogenesis, glycogen synthesis, and glycogenolysis, which can explain the reason for the relative liver weight increasing rapidly near the end of incubation. This result demonstrated that the liver, as one organ of metabolism, might play an important role in the last 2 to 3 d of incubation. It is necessary to mentioned that the small intestine, as another digestive organ, plays a key role in the avian because of its key function in absorbing nutrients. In our study, both the absolute weight and relative weight of small intestine exhibited a gradual increase throughout the hatching process with a sharp rise before hatch. Similar results were also reported by Dong et al. ( 2012b 2012b ). This change, as has also been observed in precocial poultry, such as chickens (Li et al., 2013 2013 ) and turkeys (de Oliveira et al., 2009 2009 ). A rapid increase in intestinal weight and relative weight from E15 to DOH was to prepare for enteral nutrition. In addition, the lung and thorax were classified as the fourth type of organ. During chick embryonic development, the formation of the vascular system of the lung was on E17, and the respiratory function of lung began close to hatching. The reasons why the development of lung showed such different patterns between precocial and altricial species required further investigation. To test the growth pattern of organs, correlations were observed in body weight and organ weight. In addition to the yolk, ten other organs (head, brain, heart, liver, lung, kidney, gizzard, small intestine, leg, and thorax) were positively correlated with embryo body weight. When these organ weights were plotted as a function of embryonic body weight, a logarithmic, linearly or power model was used according to the scatter diagram plot. The results indicated that the ontogeny of organs was not linear with that of the embryo. Furthermore, organs had their own special growth patterns. Gene expression 2 During the last stage of chick embryonic development, the major nutrient source for embryos not only derived from the yolk sac membrane, but also via the yolk stalk into the small intestine (Noy and Sklan, 1998 1998 ). Dong et al. ( 2012b 2012b ) had reported that nutrient absorption shifted from the sac membrane to the small intestine between E16 to DOH for pigeons. Thus, the small intestine plays a central role in nutrient absorption at the end of the incubation period. To our knowledge, the absorptive capacity of small intestine can be evaluated by studying the nutrient transporter gene expression pattern. Therefore, we investigated the gene expression of 13 nutrient transporters normally expressed in the small intestine that were responsible for nitrogen nutrient uptake. The mRNA expressions of EAAT3 , b 0,+ AT , y + LAT2 , PepT1 , LAT4 , NHE2 , and NHE3 showed similar patterns in the small intestine, with a slight increase or no change from E9 to E15 and a rapid rise from E15 to DOH, suggesting an importance in the last 2 or 3 d before hatching. According to Hirst ( 1993 1993 ), intestinal amino acid transporter expression is known to be induced by raised levels of dietary protein. This suggested that provision of nutrients via the yolk stalk from E16 to DOH may provide a nutrient signal for induction of transporter mRNA. On the contrary, the gene expressions of CAT1 , CAT2 , LAT1 , EAAT2 , SNAT1 , and SNAT2 exhibited similar profiles in the small intestine, with higher levels on E9 or E11 and lower levels toward the end of incubation; this indicated that those six genes played an important role in the second stage of incubation (from E8 to E14). At the brush border membrane, peptides and free amino acids are taken into cells by peptide and amino acid transporters. Reports from our laboratory on chicken suggested that b 0,+ AT , EAAT3 , and PepT1 , which are expressed on the brush border membrane, could be used as indicators of the small intestinal growth of chick embryos (Li et al., 2013 2013 ). In this study, the mRNA levels of those three transporters increased significantly ( P < 0.0001) with embryonic day, especially b 0,+ AT , which increased approximately 220-fold from E9 to DOH, suggesting that b 0,+ AT was more important during late embryogenesis. The b 0,+ AT is a Na + -independent amino acid transporter, with a high extracellular affinity for cationic amino acids. EAAT3 , as an excitatory amino acid transporter, has a high affinity for glutamate and aspartate. Glutamate, as an oxidative fuel source, is involved in a variety of metabolic sequences in enterocytes, increasing the amounts of energy needed to meet the needs of the rapidly growing embryos. Thus, it is reasonable that mRNA expression of EAAT3 increased significantly ( P < 0.0001) from E11 to DOH in our study. Gilbert et al. ( 2007 2007 ) found that levels of EAAT3 mRNA increased linearly ( P =0.001) from E20 to D14 in broiler small intestine, and the most important site for free amino acid assimilation was the ileum. These changes suggested that EAAT3 served as an important role in the growth of the small intestine for its key function in nutrient uptake. Amino acids are transported into the enterocyte as di-or tri-peptides through PepT1 (Leibach and Ganapathy, 1996 1996 ), which is more efficient then through the free amino acid transporters (Daniel, 2004 2004 ). Dong et al. ( 2012b 2012b ) stated that the mRNA levels of PepT1 increased linearly ( P < 0.0001) from E12 to D14 in pigeon. The mRNA levels of PepT1 were greatest in the duodenum compared with the ileum, but were of middle levels in jejunum; this was consistent with studies in chicken by Gilbert et al. ( 2007 2007 ). In this study, expression of PepT1 mRNA increased significantly from E13 to DOH, suggesting an importance in peptide transport in pigeon embryo development. In addition, the quantities of NHE2 and NHE3 expressed at the brush border membrane were also evaluated in this study. The NHE2 and NHE3 belong to the family of antiporters, known generically as Na + /H + exchangers ( NHEs ). It is well known that Na + /H + exchange is crucial for the regulation of intracellular H + in exchange for external Na + . However, Na + /H + exchange is up- and down-regulated as a part of digestion and diarrheal diseases via intestinal neutral NaCl absorption (Orlowski and Grinstein, 1997 1997 ). In our study, the mRNA expression of NHE2 and NHE3 increased significantly ( P < 0.0001) on the day of hatching, which was similar to the expression of b 0, + AT mRNA, suggesting it becomes more important in the late stage of incubation. Gene expression of the amino acid transporters on the basolateral membrane is different from that on the brush border membrane (Gilbert et al., 2007 2007 ). In our study, the genes for EAAT2 , CAT1 , CAT2 and LAT1 are expressed in the basolateral membrane with higher levels on E9 or E11, then decrease with age, suggesting an important role in pigeon embryonic growth. In the early stages of embryonic development, nutrients are absorbed into enterocytes from the blood through the basolateral membrane. Thus, the four genes may be expressed with higher levels on E9 or E11. It is well known that CAT proteins transport cationic amino acids in a Na + -independent way (Closs et al., 2006 2006 ). However, the system y + cationic amino acid transport ( CAT ) family undergoes adaptive upregulation during amino acid starvation (Humphrey et al., 2006 2006 ) and thus might be repressed by nutrient provision from the yolk stalk. Zeng et al. ( 2011 2011 ) found that the level of CAT1 in broiler intestine increased linearly with age ( P < 0.0001) but changed little during embryonic development, while CAT2 mRNA increased from E9 to E17, then decreased on DOH. In our study, the CAT1 and CAT2 exhibit similar expression patterns, peaking on E11, then decreasing gradually until the end of incubation. Gilbert et al. ( 2007 2007 ) reported similar findings in chicken, with the expressions of CAT1 and CAT2 decreasing with age. LAT1 was investigated widely on the basis of its function to transport large neutral amino acids into Xenopus oocytes when expressed with 4F2hc (Kanai et al., 1998 1998 ; Mastroberardino et al., 1998 1998 ). In our study, LAT1 expression in the intestine was at a high level on E9 and gradually decreased in expression from E11 to DOH. The sodium-coupled neutral amino acid transporter 1 (SNAT1) is a membrane protein, which plays a crucial role in the uptake of glutamine into cells in the central nervous system (Chaudhry et al., 2002 2002 ). SNAT1 is also responsible for the transport of neutral amino acids into or out of cells in mammals (McGivan and Bungard, 2007 2007 ). So far, SNAT1 is recognized as a target for oncogene action because the amount of its mRNA is increased in human hepatocarcinoma cells. In this study, the mRNA level of SNAT1 was relatively high on E9 and decreased to a low level from E11 to DOH, suggesting an important role during incubation. A similar gene expression pattern was observed in SNAT2 . Our study found that the gene expression of SNAT2 was greater than that of SNAT1 throughout development, which suggested that SNAT2 might be more important than SNAT1 in the small intestine during embryonic development. This phenomenon can be explained by the fact that SNAT2 belongs to the system A transporter and is expressed in the intestine and most likely reflects the activity of system A (Bröer, 2008 2008 ). However, SNAT2 appears adaptive upregulation during the amino acids starvation (Tanaka et al., 2005 2005 ). Therefore, the gene expression of SNAT2 might be repressed by nutrient provision from the yolk stalk. Furthermore, we demonstrated that gene expression of y + LAT2 and LAT4 increased slowly from E9 to E15, and then increased rapidly near DOH, suggesting an important role in the post-hatch pigeons. Similarly, the results for y + LAT2 were also reported in chicken to increase significantly from E18 to DOH (Gilbert et al., 2007 2007 ). A similar result for LAT4 was observed in chickens by Zeng et al. ( 2011 2011 ). To test the relationship between gene expression of nutrient transporters and embryo and small intestinal weight, we used regression analysis. Our study demonstrated that there was a strong correlation between gene expressions of EAAT3 and PepT1 and embryo weight, while there was a strong negative correlation between CAT1 gene and body weight. This suggested those three genes were more important in embryonic development. Furthermore, we found that most of those gene expressions detected in our study were strongly correlated with small intestinal weight, such as b 0,+ AT , y + LAT2 , CAT1 , LAT4 , EAAT3 , PepT1 , NHE2 , and NHE3 . This indicated gene expression of those nutrient transporters might facilitate the digestive capacity of the small intestine in pigeon during embryonic development. However, whether the expression of those genes can be regarded as indicators for the growth of the small intestine requires further research. Conclusions 1 In our study, we classified ten organs into four types according to changes in relative weight during embryonic development. These results provide a comprehensive study of amino acid and peptide transporter mRNA in the small intestine of pigeons. Future studies will involve transporter proteins and their functionality to understand nutrient absorption capacity of small intestine in the pigeon. References WK Al-Murrani . 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All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1400340 Project supported by the Spark Program of Guangdong, China (No. 2012A020603012) 2014-11-30 2015-05-13 2015-03-18 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 2015 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 16