This article was downloaded by: [FU Berlin] On: 03 July 2015, At: 07:16 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG

British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20

Polymorphism and expression of insulin-like growth factor 1 (IGF1) gene and its association with growth traits in chicken a

a

a

a

a

T.K. Bhattacharya , R.N. Chatterjee , K. Dushyanth , C. Paswan , R. Shukla & M. Shanmugam

a

a

Directorate of Poultry Research, Hyderabad, India Accepted author version posted online: 10 Jun 2015.Published online: 17 Jun 2015.

Click for updates To cite this article: T.K. Bhattacharya, R.N. Chatterjee, K. Dushyanth, C. Paswan, R. Shukla & M. Shanmugam (2015): Polymorphism and expression of insulin-like growth factor 1 (IGF1) gene and its association with growth traits in chicken, British Poultry Science, DOI: 10.1080/00071668.2015.1041098 To link to this article: http://dx.doi.org/10.1080/00071668.2015.1041098

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions

British Poultry Science, 2015 http://dx.doi.org/10.1080/00071668.2015.1041098

Polymorphism and expression of insulin-like growth factor 1 (IGF1) gene and its association with growth traits in chicken T.K. BHATTACHARYA, R.N. CHATTERJEE, K. DUSHYANTH, C. PASWAN, R. SHUKLA, M. SHANMUGAM

AND

Downloaded by [FU Berlin] at 07:16 03 July 2015

Directorate of Poultry Research, Hyderabad, India

Abstract 1. The objectives of the study were to detect polymorphism in the coding region of the IGF1 gene, explore the expression profile and estimate association with growth traits in indigenous and exotic chickens. 2. A total of 12 haplotypes were found in Cornish, control layer and Aseel breeds of chicken in which the h1 haplotype was most frequent. 3. Nucleotide substitutions among haplotypes were found at 21 positions in the IGF1 gene in which 4 substitutions resulted in non-synonymous mutations in the receptor binding domain of the IGF1 protein. 4. The haplogroup showed a significant effect on body weight at 24 and 42 d of age in the control layer line, body weight at 42 d and daily weight gain between 29 and 42 d in the control broiler line, daily weight gain between 29 and 42 d in Cornish, and body weights at 42 d as well as daily weight gain between 29 and 42 d in Aseel birds. 5. IGF1 expression varied among the breeds during embryonic and post-hatch periods. The expression among the haplogroups varied in different chicken tissues. The effect of haplogroup on myofibre number in pectoral muscle was non-significant, although there was significant variation in numbers between d 1 and d 42, and between broiler and layer lines. 6. It was concluded that the coding region of the IGF1 gene was polymorphic, expressed differentially during the pre-hatch and post-hatch periods, and haplogroups showed significant association with growth traits in chicken.

INTRODUCTION Insulin-like growth factor-1 (IGF-1) is a protein, which has similar molecular structure to insulin and plays a major role in proliferation, differentiation and metabolism of myogenic cell lines in animals including chicken (Duclos, 2005). The IGF1 gene is involved in growth of various tissues like muscle, cartilage and bones (Tirapegui, 1999). In humans, IGF1 is primarily involved in determination of height during childhood and puberty and is sometimes involved in the development of dwarfism (Blum et al., 2007). The gene also repairs damage to nerve, brain, heart and several other organs by stimulating organ synthesis (Mangiola et al., 2014). The IGF1 hormone

reduces body fat by excess use of body fat reserves as a source of energy instead of using body glucose by insulin (Arnhold et al., 2000). The chicken IGF1 gene is located on chromosome 1 and the gene length is 50 kb (Klein et al., 1996). Duclos (1998) reported a role for IGFs in regulating body and muscle growth in chickens. Researchers also reported an effect of circulating IGF1 on growth rate in chickens (Scanes et al., 1989; Ballard et al., 1990). The mRNA level in muscle was higher in a high-growth line compared with a low-growth line (Beccavin et al., 2001). The expression of IGF1 is ubiquitous, being present in several tissues including muscle, liver, kidney, heart, brain, intestine, testes and ovary (Tanaka et al., 1996; McMurtry, 1998). In a chicken line selected for increased

Correspondence to: T.K. Bhattacharya, Directorate of Poultry Research, Rajendranagar, Hyderabad 500 030, India. E-mail: [email protected] Accepted for publication 16 March 2015.

© 2015 British Poultry Science Ltd

Downloaded by [FU Berlin] at 07:16 03 July 2015

2

T.K. BHATTACHARYA ET AL.

breast yield and decreased fatness, the concentration of IGF1 in skeletal muscle was higher than in the unselected control line (Tesseraud et al., 2003). It was also reported that human IGF1 infusion in chickens enhanced growth and decreased carcass fat content (Tomas et al., 1998). Reports on polymorphism in the coding region of the IGF1 gene are scant. Amills et al. (2003) discovered a polymorphism in the promoter of the IGF1 gene in the Black Penedesenca chicken strain, which was associated with average daily gain and feed efficiency. Nagaraja et al. (2000) showed that a PstI RFLP in the 5′ flanking region of the IGF1 gene was associated with egg and egg shell weights in a White Leghorn chicken population. Abbasi and Kazemi (2011) also reported a polymorphism in the 621 bp flanking promoter and 5′ untranslated region in the fowl IGF1 gene and identified two alleles. A HinfI RFLP of a 793 bp fragment of the IGF1 gene was reported to be polymorphic in broiler chickens also indicating the presence of two alleles (Shah et al., 2012). The IGF1 gene, therefore, was selected as a candidate gene to investigate growth and conformation traits in chickens. The hypothesis was that polymorphism in the coding region of the IGF1 gene had significant effects on juvenile growth traits in chicken. The objectives of this study were therefore to detect polymorphisms in the coding region of the IGF1 gene, explore expression profiles and estimate association with growth traits in four lines of chickens.

and was used as a control to estimate genetic progress in the selected layer lines. The broiler birds were kept in the brooder house to the age of 6 weeks and then moved to the grower house. All the broiler birds were reared on a deep litter system under intensive management of farming providing ad libitum feed and water. All the layer birds were reared in a deep litter system up to 18 weeks by providing ad libitum feed and water. All the birds were hatched at the same time and housed in the same room until 18 weeks of age. The CB and Cornish chicks up to 3 weeks of age were given a feed containing 11.7 MJ metabolisable energy (ME)/kg and 210 g crude protein/kg. The CB and Cornish chicks from 3 to 6 weeks, and CL and Aseel chicks to 6 weeks were given a feed containing 10.9 MJ ME/ kg and 160 g crude protein/kg. During the brooding stage, electric heating was provided and the temperature was 32°C for week 1 with a weekly gradual decrease of 1.5°C from weeks 1 to 5. The vaccination schedule provided Marek’s Disease vaccine on d 1, Newcastle Disease vaccine on d 7, Infectious Bursal Disease vaccine on d 14 and d 24 and Newcastle Disease vaccine on d 28 to all birds. From weeks 1 to 6, 0.03 to 0.09 m2 of space for female chicks and 0.09 m2 of space for male chicks was provided in the deep-litter system. Cooling arrangements were provided during the summer season through water sprinkling on the roof and proper lighting in the shed so that birds had a congenial environment for expressing their optimum potential.

MATERIALS AND METHODS All procedures regarding housing and killing of experimental birds were approved by the Institutional Animal Ethics Committee (IAEC) of Project Directorate on Poultry, Hyderabad, India.

Experimental birds The study was conducted on one indigenous chicken breed (Aseel), two broiler breeds (control broiler and Cornish) and one layer breed (control layer) maintained at the institute farm (Hyderabad, India). The Aseel is an indigenous chicken breed of India where the body weights at 8 and 16 weeks of age were 456 and 1188 g, respectively. The control broiler (CB) line is a synthetic coloured broiler line which was randomly bred and pedigreed over the last 10 generations. The body weight of the CB population at 5 weeks of age was 625.5 ± 0.13 g (PDP Annual Report, 2010). The body weights of Cornish birds at 6 and 20 weeks of age were 668 and 1986 g, respectively (PDP Annual Report, 2012). The control layer (CL) was a randomly bred population

Sample collection and isolation Blood samples were collected from 187 birds from the CL line, 186 birds from CB, 176 Cornish birds and 174 Aseel birds. A volume of 50 μl blood was collected aseptically from the wing vein. Genomic DNA was isolated from blood cells following a standard protocol (Bhattacharya et al., 2012). The quality of DNA was verified by 0.8% agarose gel electrophoresis while quantity was checked by spectrophotometer. All DNA stocks were diluted with sterile double-distilled water to create a standard DNA concentration of 100 µg/µl. A total of 40 birds from each of control broiler, Cornish, control layer and Aseel breeds were killed at d 1, 14, 28 and 42 of age by cervical dislocation. The pectoral muscle tissue was collected from each bird for the gene expression study, with muscle kept in RNA later. For the histological study, muscle was kept in 10% formalin. For the pre-hatch gene expression study, 5 fertile eggs (chicken embryos) from each of the Aseel, Cornish, control broiler and control layer lines were collected from d 5 (E5) to d 20 (E20) of embryonic stage. The eggs were cleaned with

IGF POLYMORPHISM AND GROWTH

3

Table 1. Primers for amplification of coding regions of the chicken IGF-1 gene Primer name IGF-1E1F IGF-1E1R IGF-1E2F IGF-1E2R IGF-1E3F IGF-1E3R IGF-1E4F IGF-1E4R

Primer sequence (5′ -3′) GCT GTT TCC TGT CTA CAG TG CTT CAA GAA ATC ACA AAA GCA G GTG AAG ATG CAC ACT GTG TC TGA AGT AGA AGC CTC TGT CTC GTA AGC CTA CAG GGT ATG GAT C CTT TTG TGC TTT TGG CAT ATC AG GAA GTG CAT TTG AAG AAT ACA AG AGT CTT CCA ATG TTT AAC AAA TAA T

tissue paper soaked with 70% ethanol before use for isolation of total RNA.

Downloaded by [FU Berlin] at 07:16 03 July 2015

PCR, single-stranded conformation polymorphism and sequencing The IGF1 gene consists of 4 exons for which primers were designed from the available chicken IGF1 sequence (GenBank accession No. NC_006088) using DNASTAR software (Table 1). The PCR reaction was set up with 50 µg of DNA template, 10 ng of each primer, 1.5 mM of MgCl2, 100 µM of each dNTP, 1× assay buffer and 0.25 U of Taq DNA polymerase (MBI Fermentas, Amherst NY, USA). The PCR was performed with initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, annealing at a specific temperature (Table 1) and extension at 72°C for 30 s with a final extension at 72°C for 10 min. A 12% native PAGE (50:1, acrylamide and bisacrylamide) with 5% glycerol was prepared to resolve the SSCP pattern following a standard protocol (Bhattacharya et al., 2012). A volume of 3 µl PCR product mixed with 15 µl formamide dye (95% formamide, 0.025% xylene cyanol, 0.025% bromophenol blue, 0.5 M EDTA) was denatured at 95°C for 5 min followed by snap cooling on ice for 15 min. The product was loaded in the gel and electrophoresis was performed at 4°C for 12 h at 200 V followed by staining with silver nitrate to visualise banding patterns. Three PCR products amplified from each fragment of the IGF1 gene, derived with HotStarHiFidelity DNA Polymerase (MBI Fermentas) were sequenced with the fragment-specific primers from both ends using the automated dye– terminator cycle sequencing method provided with the ABI PRIZM 377 DNA sequencer (Perkin-Elmer, Massachusetts, USA). Haplotypes Haplotypes were constructed by combining SSCP patterns of all the fragments of the IGF1 gene of each individual bird. Haplotypes in diploid state of an individual reveal the haplogroup of that animal. Haplotype sequences were analysed with DNASTAR Software (Lasergene Inc.,

Fragment Exon 1 (309 Exon 2 (157 Exon 3 (182 Exon 4 (149

Annealing temperature (°C) 57

bp) 57 bp) 59 bp) 59 bp)

Massachusetts, USA). Frequencies of haplotype and their combinations were calculated by the gene counting method (Bhattacharya et al., 2012). RT-PCR and qPCR Total RNA was isolated from pectoral muscle following a standard TRIZOL method and firststrand cDNA was synthesised with reverse transcriptase enzyme (Bhattacharya and Chatterjee, 2013). A pair of primers, IGF1QF: 5′GATGCACACTGTGTCCTAC-3′ and IGF1QR: 5′ACGAACTGAAGAGCATCAAC-3′ were designed from the chicken cDNA sequences of the IGF1 gene (GenBank accession No. NC_006088.3) with DNASTAR software (Lasergene Inc.) for qPCR study. A 119 bp fragment of the GAPDH gene was amplified at 57°C using a pair of primers, namely QGAPDHF: 5′CTGCCGTCCTCTCTGGC-3′ and QGAPDHR: 5′GACAGTGCCCTTGAAGTGT-3′ designed from the chicken GAPDH sequence (Accession No. AF047874) with DNASTAR software (Lasergene Inc.). qPCR was performed for the IGF1 gene along with the GAPDH gene as an internal control. cDNA templates were used in a thermal cycler (Stratagene M×3000P) machine with Platinum SYBR Green qPCR UDG supermix (Invitrogen, Massachusetts, USA). The 121 bp fragment of the IGF1 gene was amplified at an annealing temperature of 57°C. Reactions were prepared in triplicate with a final volume of 25 µl containing 12.5 µl of Platinum SYBR Green qPCR supermix, 0.5 µl of ROX reference dye, 0.2 µM of each primer and 2 µl of cDNA. The qPCR conditions involved initial denaturation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 1 min and extension at 72°C for 30 s. Following amplification, a dissociation melting curve analysis was conducted with programming the PCR machine from 55 to 95°C to detect possible non-specific products. Fluorescence threshold was determined by the default method at 32.5% with Stratagene software for the M×3000P real-time PCR machine. The Ct values of each sample were noted and average Ct values of each sample generated in triplicate qPCR reactions were used in calculating the fold

4

T.K. BHATTACHARYA ET AL.

change (fold change = 2−ΔΔCt) of gene expression at different ages of birds.

RESULTS Polymorphisms

Growth traits Body weights at d 1, 14, 28 and 42 were measured for all the birds. Growth rates between d 1 and d 14, d 14 and d 28, and d 28 and d 42 were calculated for all the birds. At 6 weeks of age, 20 birds (10 males and 10 females) from each line were killed by cervical dislocation.

Downloaded by [FU Berlin] at 07:16 03 July 2015

Histological examination The pectoral muscle tissue was collected from 40 birds from each of the control broiler, Cornish, control layer and Aseel lines used in this study on d 1 and 42 of age, and kept in 10% formalin overnight. The tissue was kept under running water. The tissue was treated with ethanol and xyline for 9 h and embedded in paraffin wax for 2 h followed by freezing at −10°C overnight. The muscle was cross-sectioned perpendicular to the direction of the myofibres. Tissue sections of 10 μm were prepared in a microtome and kept for drying at 40°C for 2 to 3 h. The slide was stained with haematoxylin and eosin and mounted with DPX. The slide was observed on 10 areas, randomly chosen in the slide, under an inverted fluorescent microscope where myofibres were viewed and counted at 60× magnification. Photographs were used for counting myofibre numbers. The myofibre numbers were counted in 2 × 2 cm regions in each of the four corners and in the centre of the photographs in 10 random locations on each slide and an average taken. Statistical analysis The association of haplogroup and traits was estimated following the least-square maximum likelihood method of the LSML90 package (Harvey, 1991), where haplotype and sex were used as fixed effects and sire as random effect. Thus, the model used for this analysis was Yijklm = µ + Si + Bj + HPLk + BXHPLjk + eijkl, where µ is the overall mean; Si, the effect of the ith sire; Bj, the effect of the jth sex; HPLk, the effect of the kth haplotype; BHPLjk, the interaction of sex and haplotype; and eijkl, the random error assumed to be normally and independently distributed with a mean of 0 and a standard deviation of σ2e. The effects of gene expressions on growth traits were also estimated by the linear regression technique using SPSS software. The effect of haplogroups on number of myofibres was estimated using ANOVA, where haplogroup and sex were used as fixed effects and sire as a random effect.

A total of 12 haplotypes were found in Cornish, control layer and Aseel breeds of chicken (Table 2). In control broilers, all the haplotypes except h8 were observed. Of all the haplotypes, h1 had the highest frequency in all the lines, whereas the lowest frequency was found for the h8 haplotype (0.001) in control layer birds, h11 and h12 in control broiler (0.005) and Cornish (0.005) lines and h5 in Aseel chicken (0.01). Consequently, a total of 12 haplogroups were found in all the lines except control broilers where the h1h8 combination was absent (Table 3). In all the lines, the h1h1 haplogroup was the most frequent haplogroup, whereas the least frequent haplogroup was h1h11 and h1h12 (0.005) in control layers, h1h11 (0.005) in control broiler, h1h7 (0.01) in Cornish birds and h1h5, h1h9 and h1h10 (0.02) in Aseel birds.

Table 2.

Haplotype frequency in different chicken lines

Control layer Haplotypes (n = 187) h1 h2 h3 h4 h5 h6 h7 h8 h9 h10 h11 h12

0.77 0.03 0.09 0.02 0.005 0.005 0.03 0.001 0.02 0.01 0.002 0.002

Control broiler (n = 186) 0.68 0.09 0.06 0.05 0.05 0.01 0.008 – 0.03 0.04 0.005 0.005

Cornish Aseel (n = 176) (n = 174) 0.57 0.06 0.05 0.06 0.03 0.05 0.005 0.03 0.02 0.01 0.08 0.03

0.57 0.07 0.06 0.08 0.01 0.07 0.05 0.02 0.01 0.01 0.02 0.03

n is the number of birds in each breed group.

Table 3.

Haplogroups h1h1 h1h2 h1h3 h1h4 h1h5 h1h6 h1h7 h1h8 h1h9 h1h10 h1h11 h1h12

Haplogroup frequency in the chicken lines Control layer (n = 187)

Control broiler (n = 186)

0.53 0.08 0.16 0.05 0.02 0.01 0.06 0.01 0.04 0.02 0.005 0.005

0.35 0.19 0.12 0.09 0.09 0.02 0.02 – 0.05 0.05 0.005 0.01

n is the number of birds in each breed group.

Cornish Aseel (n = 176) (n = 174) 0.15 0.12 0.10 0.12 0.06 0.09 0.01 0.05 0.04 0.02 0.17 0.07

0.13 0.14 0.12 0.16 0.02 0.13 0.10 0.05 0.02 0.02 0.05 0.06

IGF POLYMORPHISM AND GROWTH

Downloaded by [FU Berlin] at 07:16 03 July 2015

Sequence variability The nucleotide substitutions were found at 21 positions in the haplotypes of the IGF1 gene. Of the 21 substitutions, 3 substitutions were found in the 5′UTR region (Supplementary Figure 1). Two substitutions which were present in the signal peptide of the pre-protein were synonymous. A total of 7 nucleotide substitutions were found in the receptor binding domain of the peptide in which nucleotide substitutions at positions 539, 542, 567 and 594 resulted in non-synonymous mutations affecting amino acid changes in the IGF1 protein (Supplementary Figure 2). In the E-domain of the peptide, 5 nucleotide substitutions were found of which 3 substitutions at positions 611, 617 and 702 showed non-synonymous substitutions in the mature protein. In the 3′UTR region of the gene, 4 nucleotide substitutions were observed. Sequences of haplotypes were submitted to the NCBI Genbank database under accession numbers JN593011, JN593012, JN593013, JN593014, JN593015, JN593016, JN593017, JN593018, JN609548, JN609549, JN609550 and JN609551 for h1, h2, h3, h4, h5, h6, h7, h8, h9, h10, h11 and h12 haplotypes, respectively.

Association of haplotype with trait The haplogroup showed a significant effect (P < 0.05) on body weight at 28 and 42 d of age in the control layer line, body weight at 42 d (Table 4) and daily weight gain between 29 and 42 d in the control broiler line (Table 5), daily weight gain between 29 and 42 d in Cornish birds (Table 5), and body weight at 42 d (Table 4) as well as daily weight gain between 29 and 42 d in Aseel birds (Table 5). In the control layer line, the Table 4. bw1d (g) Haplogroup h1h1 h1h2 h1h3 h1h4 h1h5 h1h6 h1h7 h1h8 h1h9 h1h10 h1h11 h1h12 RSD

5

highest body weight at 28 d was found in h1h8 and h1h9 haplogroups, whereas the lowest body weight was found in the h1h12 haplogroup. The h1h8 and h1h9 haplotype combinations had 38% higher body weight than the h1h2 group. The trend of body weight at 28 d among haplogroups was h1h8 and h1h9 > h1h1, h1h10, h1h2, h1h3, h1h5 and h1h7 > h1h11, h1h4 and h1h6. At d 42, the highest body weight was found in the h1h9 haplogroup, and the lowest body weight was observed in the h1h6 haplogroup. The h1h9 group showed 37.8% higher body weight than the h1h6 haplogroup. The trend of performance among haplogroups was h1h9 > h1h1, h1h2, h1h3, h1h4, h1h5, h1h7, h1h8, h1h9, h1h10, h1h11 and h1h12 > h1h6. In the control broiler line, the highest body weight at d 42 was found in the h1h11 haplogroup, whereas the lowest performance was observed in the h1h12 haplogroup. The h1h11 birds revealed 14.9% higher body weight at d 42 than the h1h12 haplogroup. The trend on performance among haplogroups was h1h11, h1h10, h1h1 and h1h6 > h1h3, h1h4, h1h5 and h1h9 > h1h12, h1h2 and h1h7. For daily weight gain between 29 and 42 d, the h1h11 haplogroup performed the best (39.0 ± 9.3 g/d), whereas the h1h7 group performed at the lowest level (26.03 ± 5.69 g/d). The h1h11 birds showed 49.8% superiority in daily gain over h1h7 birds. The trend in performance among haplogroups was h1h11, h1h1 and h1h10 > h1h2, h1h4, h1h9, h1h3 and h1h5 > h1h12 and h1h7. In the Cornish breed, daily body weight gain between 29 and 42 d was found at the highest level (29.88 ± 5.41 g/d) in the h1h1 haplogroup, whereas the lowest gain was observed in the h1h9 group. The h1h1 group showed 74.9% superiority over the h1h9 group for this

Haplogroup-wise mean body weight in the chicken lines bw14d (g)

bw28d (g)

bw42d (g)

CL

CB

CR

ASL

CL

CB

CR

ASL

CL

CB

CR

ASL

CL

CB

CR

ASL

34.9 33.5 35.1 32.8 35.9 34.5 36.8 38.8 35.8 36.3 37.2 36.2 1.4

44.5 39.1 39.3 40.4 39.5 41.0 38.6 – 41.2 39.6 39.3 39.7 8.7

41.5 41.1 42.0 41.8 40.6 41.4 34.5 39.4 39.7 43.5 40.4 40.4 1.2

33.7 35.1 32.5 34.3 31.2 36.4 32.4 32.4 36.4 30.5 34.0 32.1 1.2

65.1 68.1 68.3 61.2 66.7 69.7 62.2 71.5 72.0 67.1 66.5 67.0 5.2

117.7 108.4 112.7 114.2 121.1 103.0 119.7 – 125.7 134.6 110.0 131.0 8.1

138.1 144.0 139.6 139.8 134.5 139.5 121.0 147.2 123.6 140.2 145.5 130.6 7.3

66.7 74.0 63.4 64.7 70.5 72.4 57.6 69.5 65.5 60.0 68.7 58.9 6.1

127.5ab 130.2ab 129.9ab 116.3a 120.7ab 119.1a 125.6b 143.8c 143.6c 121.5ab 112.0a 104.0a 11.8

365.1 366.5 352.6 380.7 361.7 352.9 418.1 – 380.9 394.4 345.1 373.4 32.5

339.7 399.2 386.5 395.6 402.7 396.4 426.2 408.0 402.0 364.7 388.3 371.1 38.2

146.8 159.9 129.3 141.8 177.9 148.3 108.6 147.8 128.9 138.5 149.4 133.7 15.8

224.4b 230.3b 224.5b 229.8b 227.5b 186.5a 225.2b 230.0b 256.9c 221.2 b 225.0 b 193.0a 23.3

851.6bc 804.1a 816.7b 832.8b 824.9b 876.3bc 782.6a – 838.2b 886.4c 891.1c 775.6a 41.7

752.1 700.0 700.6 711.7 736.3 762.3 699.0 742.6 641.1 744.5 723.3 677.5 41.6

302.1c 293.9bc 259.7b 259.3b 294.7bc 265.6b 199.0a 310.10c 249.3b 225.1a 265.7b 279.1b 32.3

Within a column, values not sharing a common superscript letter are significantly different (P ≤ 0.05). bw1d, body weight at d 1; bw14d, body weight at d 14; bw28d, body weight at d 28; bw42d, body weight at d 42. CL, control layer line; CB, control broiler line; CR, Cornish breed; ASL, Aseel breed. The number of birds studied in CL, CB, CR and ASL were 187, 186, 176 and 174, respectively, in the all age groups.

a–c

6

T.K. BHATTACHARYA ET AL.

Table 5. Haplogroup wide daily body gains in the chicken lines dwg1–14d (g/d)

dwg15–28d (g/d)

dwg29–42d (g/d)

Haplogroup

CL

CB

CR

ASL

CL

CB

CR

ASL

CL

CB

CR

ASL

h1h1 h1h2 h1h3 h1h4 h1h5 h1h6 h1h7 h1h8 h1h9 h1h10 h1h11 h1h12 RSD

2.1 2.4 2.3 2.0 2.2 2.5 1.8 2.3 2.5 2.2 2.0 2.2 0.4

5.2 4.9 5.2 5.2 5.8 4.4 5.7 – 6.0 6.7 5.0 6.5 0.8

6.9 7.3 6.9 6.9 6.7 7.0 6.1 7.7 5.9 6.9 7.5 6.4 0.6

2.3 2.7 2.2 2.1 2.8 2.5 1.7 2.6 2.0 2.1 2.4 1.9 0.4

4.5 4.4 4.3 3.9 3.8 3.5 4.5 5.1 5.1 3.8 3.2 2.6 0.8

17.6 18.4 17.1 19.0 17.1 17.8 21.3 – 18.2 18.5 16.7 17.3 2.1

12.9 18.2 17.6 18.2 19.1 18.3 21.8 18.6 19.8 16.0 17.3 17.1 5.7

5.7 6.1 4.7 5.5 7.6 5.4 3.6 5.5 4.5 5.6 5.7 5.3 0.8

6.9 7.1 6.7 8.1 7.6 4.8 7.1 6.1 8.0 7.1 8.0 6.3 1.9

34.7bc 31.2ab 33.1ab 32.2ab 33.0ab 37.3bc 26.0a – 32.6ab 35.1bc 39.0c 28.7a 4.6

29.8b 21.4a 22.4ab 22.5ab 23.8ab 26.1b 19.4a 23.8ab 17.0a 27.1b 23.9ab 21.8a 7.5

11.0c 9.5bc 9.3bc 8.3b 8.3b 8.3b 6.4a 11.5c 8.6b 6.1a 8.3b 10.3c 1.6

Within a column, values not sharing a common superscript letter are significantly different (P ≤ 0.05). dwg1–14, daily body weight gain between d 1 and d 14; dwg15–28d, daily body weight gain between d 15 and d 28; dwg29–42d, daily body weight gain between d 29 and d 42. CL, control layer line; CB, control broiler line; CR, Cornish breed; ASL, Aseel breed. The number of birds studied in CL, CB, CR and ASL were 187, 186, 176 and 174, respectively, in the all age groups.

IGF1 expression at the embryonic stage

trait. In the Aseel breed, the highest body weight at 42 d was found in the h1h8 haplogroup, whereas the h1h7 haplogroup had the lowest body weight. The h1h8 haplotype had 55.8% superiority over h1h7 group. The performance trend of the haplogroups for the trait was h1h8, h1h1, h1h2 and h1h5 > h1h11, h1h12, h1h3, h1h4 and h1h6 > h1h10 and h1h7. The daily body weight gain between 29 and 42 d was the highest in the h1h8 haplogroup, whereas the lowest daily gain was found in the h1h10 haplogroup. The h1h8 birds showed 87.4% superiority over the h1h10 birds. The performance trend among haplogroups was h1h8, h1h12, h1h1, h1h2 and h1h3 > h1h11, h1h4, h1h5, h1h6 and h1h9 > h1h10 and h1h7.

43 42 41 40 Fold change

Downloaded by [FU Berlin] at 07:16 03 July 2015

a–c

39 38 37

Expression of the IGF1 gene in the breast muscle of Aseel, control layer, Cornish and control broiler birds was quantified during d 5 to d 20 of embryonic stage by qPCR. The ΔCt values of all the breeds were pooled for determining the expression of this gene during each embryonic stage. The highest expression of the gene was observed on d 5 (E5), whereas the lowest expression was found on d 10 (E10) of embryonic development (Figure 1). Expression of the IGF1 gene decreased gradually from d 5 to d 10 and again increased up to d 13. From d 13 to d 17, expression of the IGF1 in breast muscle again decreased, and from this stage it increased up to d 20. It is

c bc

b

b

ab a

ab

a

b

ab ab a

a

ab

a

a

36 35 34 33

Figure 1. IGF-1 expression profile during the embryonic stage in chicken. Different labels indicate significant differences of IGF1 expression at P < 0.05. The error bars indicate SEM.

Fold change

IGF POLYMORPHISM AND GROWTH 35.0 34.5 34.0 33.5 33.0 32.5 32.0 31.5 31.0 30.5 30.0

c

c

7

Table 6. Haplogroup-wise expression of IGF1 in breast muscle of chicken

b

Fold change, ΔCT Haplogroup a

Day 1

Day 14

Day 28

Day 42

Figure 2. Post-hatch expression profile of IGF-1 gene in chicken. Different labels indicate significant differences of IGF1 expression among birds of 4 age groups, that is, d 1, d 14, d 28 and d 42. The error bars indicate SEM.

h1h1 h1h2 h1h3 h1h4 h1h5 h1h6 h1h7 h1h8 h1h9 h1h10 h1h11 h1h12

Day 1 34.1 36.4 31.2 33.4 31.2 25.6 31.4 34.3 37.0 34.3 28.1 28.9

± ± ± ± ± ± ± ± ± ± ± ±

Day 14 b

4.6 3.3c 4.2ab 4.1b 4.0ab 4.2a 2.8ab 3.1b 3.7c 3.9b 4.1a 2.3a

32.9 35.3 32.1 33.9 30.9 25.2 31.3 31.1 35.3 34.4 30.4 26.1

± ± ± ± ± ± ± ± ± ± ± ±

Day 28 b

2.6 3.9c 2.7b 3.1bc 2.6b 3.0a 3.5b 2.9b 2.4c 3.8c 4.0b 4.2a

33.2 35.0 34.0 34.3 30.8 27.3 33.8 36.0 37.0 36.0 32.8 30.1

± ± ± ± ± ± ± ± ± ± ± ±

Day 42 bc

3.2 2.5bc 3.3bc 2.9bc 4.1b 3.4a 2.8bc 2.6c 3.1c 3.0c 2.7b 2.0b

31.0 33.3 32.7 31.0 28.7 27.1 33.8 32.8 35.8 32.6 27.5 27.8

± ± ± ± ± ± ± ± ± ± ± ±

3.4b 2.7bc 3.3b 1.9b 2.4a 3.2a 1.8bc 4.2b 3.9c 2.8b 3.1a 3.4a

a–c

Downloaded by [FU Berlin] at 07:16 03 July 2015

clear that the IGF1 expression in muscle varied at different embryonic stages. IGF1 expression post-hatch IGF1expression was analysed on d 1, 14, 28 and 42 after hatching in control layer, control broiler, Cornish and Aseel birds. The ΔCt values of all the breeds were pooled to determine the expression profile during the juvenile stage. Expression in muscle was highest on d 1 and d 28, whereas the lowest expression occurred on d 42 (Figure 2). However, the expression pattern in muscle varied significantly (P < 0.05) among different age groups. Haplogroup expression The haplogroups showed a significant (P < 0.05) association with IGF1 expression in the breast muscle. The h1h2 and h1h9 haplogroups had higher IGF1 expression in muscle than other haplogroups in all 4 age groups, whereas the h1h6 group showed the lowest expression (Table 6). However, other haplogroups had variable magnitudes of expression, which were observed to be significantly different among each other. Effect on myofibres The effect of haplogroups on myofibre number was non-significant (P < 0.05), although birds with the h1h6 haplogroup showed apparently higher number of myofibres (2.85/4 cm2) than other haplogroups (Table 7). Among the lines, there were non-significant differences both at d 1 and d 42 between the broiler and layer birds (Figure 3). There were also non-significant differences in myofibre numbers between male and female birds (2.37 vs. 1.85). In both broiler (1.34 vs. 2.90) and layer (1.22 vs. 2.46) lines,

Within a column, values not sharing a common superscript letter are significantly different (P ≤ 0.05). ΔCT indicates change in CT values of IGF1 expression in muscle.

Table 7. Haplogroup-wise myofibre number in breast muscle of chicken (mean ± SE)a Haplogroup h1h1 h1h2 h1h3 h1h4 h1h5 h1h6 h1h7 h1h8 h1h9 h1h10 h1h11 h1h12

Number of birds 45 21 19 16 7 11 8 5 7 5 10 6

Average number of myofibres ± SE 1.929 2.596 2.117 1.700 2.233 2.850 2.250 1.733 1.100 1.533 2.300 2.110

± ± ± ± ± ± ± ± ± ± ± ±

0.086 0.104 0.101 0.175 0.143 0.175 0.152 0.143 0.152 0.143 0.248 0.186

a The myofibre number did not differ significantly (P < 0.05) among haplogroups.

the numbers of myofibres were significantly higher in birds at d 42 than at d 1.

DISCUSSION A total of 12 haplotypes were observed in the IGF1 coding region across the 4 chicken lines but the h8 haplotype was absent in the control broiler line. In all the lines, the most predominant haplotype was h1, but the least frequent haplotype varied from line to line. The frequencies of other haplotypes also varied among the lines: haplotypes such as h5, h6, h11 and h12 in control layers; h7, h11 and h12 in control broilers; and h7 in Cornish birds were of a novel type possessing very low frequency of less than 1% in the population. In the Aseel breed, there was an absence of any novel haplotypes. Shah et al. (2012) reported PCR-RFLP of a 793 bp

8

T.K. BHATTACHARYA ET AL.

Day 1

1a

2a

Day 1

Downloaded by [FU Berlin] at 07:16 03 July 2015

1b

2b

Day 42

1a

2a

Day 42

1b

2b

Figure 3. Cross section of breast muscle at different ages in control line broiler (CB) and control line layer (CL) chicken. 1a, CB male; 1b, CB female; 2a, CL male; 2b, CL female. The figure shows myofibres in breast muscle of chicken (H&E, 60× magnification).

fragment of the IGF1 gene, revealing the presence of SNPs at the region determining only two alleles in a broiler chicken line. Similar observations have also been reported by Amills et al. (2003) in the Black Penedesenca chicken strain. From the present study, it is suggested that these differential haplotype distributions indicate line specificity and uniqueness of genetic architecture. The haplotype composition reveals the presence of several SNPs at different locations within the coding region as well as 5′UTR and 3′UTR regions of the gene. In the 5′UTR region, there were only three SNPs which were of a transvertional type. Amills et al. (2003) identified one SNP in the 5′UTR of Black Penedesenca chickens.

Shah et al. (2012) determined that IGF1 was mostly linked to post-hatch development. Tomas et al. (1998) demonstrated that human recombinant IGF1 significantly increased growth rate in chicken. Likewise, hepatic IGF1 mRNA levels were also shown to be higher in a high-growth strain as compared with a low-growth strain (Beccavin et al., 2001). The 5′UTR, although not contributing towards the composition of the protein, plays a significant role in post-transcriptional splicing of the mRNA, ultimately leading to the availability of mature mRNA at the nucleus. One of the important factors of protein expression is the rate of splicing of mRNA to mature mRNA which further participates in translation. The nucleotide polymorphism plays a large

Downloaded by [FU Berlin] at 07:16 03 July 2015

IGF POLYMORPHISM AND GROWTH

contribution in this splicing process, ultimately regulating protein expression. A polymorphism in the coding region may interfere with the composition of the protein if the polymorphism is of a non-synonymous type. In the present study, 14 SNPs were identified in the coding region of which 4 SNPs produced change of amino acids in the receptor binding domain of the protein. Besides, three non-synonymous types of SNPs were also found in the E-domain of the peptide. The amino acid changes in the receptor binding domain may have an effect on binding efficiency of the receptor, which ultimately exerts the biological function of the IGF1 protein in the cell. The E-domain region of the protein had amino acid changes at three locations of the protein, which may not have a direct role in receptor binding activity, but may play an important role during proteolytic cleavage of the peptide to form the mature functional peptide in terms of receptor binding domain in the IGF1 protein. There were 4 SNPs at the 3′UTR: the 3′UTR is important in the splicing activity of pre-mRNA and is thus involved in protein expression. Polymorphisms in this region may therefore play a major role in splicing activity of the pre-mRNA, ultimately affecting protein expression. The haplogroup-wise expression profile was examined in muscle for the juvenile period, particularly at d 1, 14, 28 and 42. The expression of the haplogroups varied among different age groups in birds. Of all the haplogroups, h1h2 and h1h9 had the higher expression in muscle of birds among all age groups. Further, the pre-hatch expression pattern of this gene was also analysed from d 5 to d 20 during the embryonic period. The expression patterns were different in different lines. The highest pre-hatch IGF1 expression was found in d 5 embryos, whereas the lowest expression of this gene was observed in d 10 embryo. Study of prehatch IGF1 expression is important because of its role in the development process of tissues/organs during the embryonic stage. Most of the organs are developed during the embryonic stage and become functional during post-hatch. However, the pre-hatch organ development will ensure the performance of birds during the post-hatch period. Different haplogroups performed in different ways on growth traits in different lines. In slow growing lines such as the control layer line, a significant haplogroup and trait relationship was found at d 28 and d 42 with body weight and daily body weight gain between d 29 and d 42. In another slow growing line, Aseel, only body weight at d 42 was significantly influenced by haplogroups. In fast growing lines like the control broiler line, d 42 body weight and daily weight gain between d 29 and d 42 were significantly affected by the haplogroups, whereas in another fast growing line, Cornish, only daily gain between

9

d 29 and d 42 was observed as significant for haplogroup-wise variation. The similar trend of association between IGF1 polymorphism and growth rates was observed by Gouda and Essawy (2010) in Egyptian chicken breeds. However, the differential performance of the haplogroups in different lines was genetically influenced by the genotypic pattern of genes. Unlike growth traits, myofibre numbers were not found to be significantly affected by IGF1 haplogroups, although significant differences of myofibre numbers were obtained between sexes and two age groups (d 1 and d 42). However, the high performing haplogroups for body weight may be favoured during selection of breeding sires and dams. These elite birds can improve the performance in the progeny generation. Thus, incorporating haplogroup as one of the criteria for selection of birds may rapidly improve the performance of birds even if the birds are selected at a very early age only by haplotyping of birds. In conclusion, the coding region of the IGF1 gene was polymorphic and was expressed differentially among haplogroups during the embryonic and post-hatch periods. Haplogroup showed significant association with growth traits in the chicken lines investigated.

FUNDING Authors are thankful to the Indian Council of Agricultural Research for providing financial support to carry out research works under the National Fellow project.

SUPPLEMENTAL DATA Supplemental data for this article can be accessed: http://dx.doi.org/10.1080/00071668.2015.1041098.

REFERENCES ABBASI, H.A. & KAZEMI, M. (2011) Detection of polymorphism at the insulin like growth factor-I gene in Mazandaran native chicken using polymerase chain reaction-restriction fragment length polymorphism method. American Journal of Animal and Veterinary Sciences, 6: 80–83. doi:10.3844/ ajavsp.2011.80.83 AMILLS, M., JIMENEZ, N., VILLALBA, D., TOR, M., MOLINA, E., CUBILO, D., MARCOS, C., FRANCESCH, A., SANCHEZ, A. & ESTANY, J. (2003) Identification of three single nucleotide polymorphisms in the chicken insulin-like growth factor 1 and 2 genes and their associations with growth and feeding traits. Poultry Science, 82: 1485–1493. doi:10.1093/ps/82.10.1485 ARNHOLD, I.J., OLIVEIRA, S.B., OSORIO, M.G., CARRILHO, A.J., NICOLAU, W., BIANCO, A.C. & MENDONCA, B.B. (2000) Lack of reduction in body fat after treatment with insulin-like growth factor-I in two children with growth hormone gene deletions. Journal of Endocrinological Investigation, 23: 258–262. doi:10.1007/BF03343719 BALLARD, F.J., JOHNSON, R.J., OWENS, P.C., FRANCIS, G.L., UPTON, F.M., MCMURTRY, J.P. & WALLACE, J. (1990) Chicken insulinlike growth factor-I: Amino acid sequence, radioimmunoassay, and plasma levels between strains and during growth.

Downloaded by [FU Berlin] at 07:16 03 July 2015

10

T.K. BHATTACHARYA ET AL.

General and Comparative Endocrinology, 79: 459–468. doi:10.1016/0016-6480(90)90076-X BECCAVIN, C., CHEVALIER, B., COGBURN, L.A., SIMON, J. & DUCLOS, M.J. (2001) Insulin-like growth factors and body growth in chickens divergently selected for high or low growth rate. Journal of Endocrinology, 168: 297–306. doi:10.1677/ joe.0.1680297 BHATTACHARYA, T.K. & CHATTERJEE, R.N. (2013) Polymorphism of the myostatin gene and its association with growth traits in chicken. Poultry Science, 92: 910–915. doi:10.3382/ps.201202736 BHATTACHARYA, T.K., CHATTERJEE, R.N. & PRIYANKA, M. (2012) Polymorphisms of Pit-1 gene and its association with growth traits in chicken. Poultry Science, 91: 1057–1064. doi:10.3382/ ps.2011-01990 BLUM, J.W., ELSASSER, T.H., GREGER, D.L., WITTENBERG, S., DE VRIES, F. & DISTL, O. (2007) Insulin-like growth factor type1 receptor down-regulation associated with dwarfism in Holstein calves. Domestic Animal Endocrinology, 33: 245–268. doi:10.1016/j.domaniend.2006.05.007 DUCLOS, M.J. (1998) Regulation of chicken muscle growth by insulin-like growth factors. Annals of the New York Academy of Sciences, 839: 166–171. doi:10.1111/j.1749-6632.1998. tb10752.x DUCLOS, M.J. (2005) Insulin-like growth factor-I (IGF-1) mRNA levels and chicken muscle growth. Journal of Physiology and Pharmacology, 56 (Suppl 3): 25–35. GOUDA, E.M. & ESSAWY, G.S. (2010) Polymorphism of insulinlike growth factor I gene among chicken breeds in Egypt. Zeitschrift für Naturforschung, C65: 284–288. HARVEY, W.R. (1991) User’s Guide for LSMLMW, Mixed Model Least Squares and Maximum Likelihood Computer Programme. (Columbus, OH: Ohio State University (Mimeograph)). KLEIN, S., MORRICE, D.R., SANG, H., CRITTENDEN, L.B. & BURT, D. W. (1996) Genetic and physical mapping of the chicken IGF1 gene to chromosome 1 and conservation of synteny with other vertebrate genomes. Journal of Heredity, 87: 10–14. doi:10.1093/oxfordjournals.jhered.a022946 MANGIOLA, A., VIGO, V., ANILE, C., DE BONIS, P., MARZIALI, G. & LOFRESE, G. (2014) Role and importance of IGF-1 in traumatic brain injuries. BioMed Research International, Article ID 736104.

MCMURTRY, J.P. (1998) Nutritional and developmental roles of insulin-like growth factors in poultry. Journal of Nutrition, 128: 302–305. NAGARAJA, S.C., AGGREY, S.E., YAO, J., ZADWORNY, D., FAIRFULL, R. W. & KUHNLEIN, U. (2000) Brief communication. Trait association of a genetic marker near the IFG-I gene in egglaying chickens. Journal of Heredity, 91: 150–156. doi:10.1093/jhered/91.2.150 PDP ANNUAL REPORT. (2010) PDP Annual Report 2009–2010, Project Directorate on Poultry, Hyderabad, India, p. 12. PDP ANNUAL REPORT. (2012) PDP Annual Report 2011–2012, Project Directorate on Poultry, Hyderabad, India, pp. 4–6. SCANES, C.G., DUNNINGTON, E.A., BUONOMO, F.C., DONOGHUE, D. J. & SIEGEL, P.B. (1989) Plasma concentrations of insulin like growth factors (IGF1 and IGFII) in dwarf and normal chickens of high and low eight selected lines. Growth Development and Aging, 53: 151–157. SHAH, T., DESHPANDE, S., DASGUPTA, U., SINGH, K.M. & JOSHI, C.G. (2012) Genotypic and allelic frequencies of IGF1 and IGF2 genes in broilers analysed by using PCR-RFLP techniques. Iranian Journal of Applied Animal Science, 2: 357–360. TANAKA, M., HAYASHIDA, Y., SAKAGUCHI, K., OHKUBO, T., WAKITA, M., HOSHINO, S. & NAKASHIMA, K. (1996) Growth hormone independent expression of insulin-like growth factor 1 messenger ribonucleic acid in extra hepatic tissues of the chicken. Endocrinology, 137: 30–34. TESSERAUD, S., PYM, R.A., BIHAN-DUVAL, E.L. & DUCLOS, M.J. (2003) Response of broilers selected on carcass quality to dietary protein supply: Live performance, muscle development, and circulating insulin-like growth factors (IGF-I and -II). Poultry Science, 82: 1011–1016. doi:10.1093/ps/ 82.6.1011 TIRAPEGUI, J. (1999) Effect of insulin-like growth factor-1 (IGF-1) on muscle and bone growth in experimental models. International Journal of Food Sciences and Nutrition, 50: 231–236. doi:10.1080/096374899101102 TOMAS, F.M., PYM, R.A., MCMURTRY, J.P. & FRANCIS, G.L. (1998) Insulin-like growth factor (IGF)-I but not IGF-II promotes lean growth and feed efficiency in broiler chickens. General and Comparative Endocrinology, 110: 262–275. doi:10.1006/ gcen.1998.7072

Polymorphism and expression of insulin-like growth factor 1 (IGF1) gene and its association with growth traits in chicken.

1. The objectives of the study were to detect polymorphism in the coding region of the IGF1 gene, explore the expression profile and estimate associat...
324KB Sizes 1 Downloads 8 Views