New strategies such as marker-gene assisted selection (MAS or GAS) provide high accuracy in animal breeding. This is necessary as demand for animal protein increases (Ranjbari et al. 2012; Gholibeikifard et al. 2013). Sheep of native breeds are a suitable source of protein in many countries. But there have been many problems, such as inaccuracy in pedigrees and farm records, leading to inefficient selection programs. To improve genetic gain and production, molecular markers associated with economic traits may be useful. The gene for insulin-like growth factor 1 (IGF1) may play important roles in the growth of multiple tissues, including mammalian muscle. Therefore it has been proposed as a candidate gene for growth traits in farm animals. It could be used in marker-assisted-selection (Honarvar et al. 2012). IGF is important in mammalian growth regulation, development and metabolism. It is made in the liver (Gholibeikifard et al. 2013; Munoz et al. 2011). Its two forms, IGF1 and IGF2 are found in most tissues. They are similar in structure and function but IGF2 is less effective than IGF1. Growth hormone (GH) cannot control IGF2 secretion. Also, the liver is the largest source of IGF found in the circulation. It has been suggested that alongside regulation by GH, nutrition might very strongly affect IGF. Nutrition has been shown to have direct and indirect effects on the IGF system (Cianfarani et al. 2005). Thus IGF-regulated physical growth is related to nutrition. IGF plays a key role in lactation, reproduction, fetal development and growth (Adam et al. 2000; Shen et al. 2003). Polymorphism in IGF1 is associated with ovine fertility traits (He et al. 2012). In cattle, IGF1 is associated with reproduction and early and late growth traits, suggesting that this gene encodes a hormone like polypeptide (Grossi Ddo et al. 2015). Salvetti et al. (2007) suggested an important role for IGF1 in the regulation of folliculogenesis and also in the pathogenesis of bovine cystic ovarian disease. IGF1 is a major gene associated with growth traits. Its product IGF1 stimulates myogenesis, participates in the activation of cell cycle genes, inhibits apoptosis, increases glucose absorbency, increases lipid synthesis, stimulates the production of progesterone in granular cells, participates in the synthesis of DNA, RNA and proteins and in cell proliferation (Chelongar et al. 2014; Ge et al. 2001). In Iran, sheep are raised mainly for meat production but around the world, sheep are raised for different causes (Ghafouri-Kesbi et al. 2008). The Zandi is native to Iran, a medium sized dual purpose breed (meat and pelt) kept in the central region. Its origin can be traced to the Karakul sheep of Shiraz (Khojasteh et al. 2006). Due to its fat tail it is suitable to lowland areas. It is reared under a migratory system. Its population is approximately two million. There is considerable interest in this breed for genetic improvement (Ghafouri-Kesbi et al. 2008). The aim of this study was to investigate IGF1 polymorphism, seeking association with early growth traits.
MATERIALS AND METHODS
120 male and female native pure Zandi lambs were selected randomly at a breeding station in Qom province. Lambing of this station occurred during late January to early February. The average weaning age was three months. Whole blood was collected from the jugular vein into vacuum tubes containing 7.5 mg ethylenediaminetetraacetic acid (EDTA), then stored at 4 ˚C. Genomic DNA was extracted from 500 μL of blood using a modified salting-out method, then stored at -20 ˚C. DNA quality and quantity were investigated on 0.8% agarose gels, comparing with a DNA ladder.
A 265 bp fragment of the ovine IGF1 gene was amplified. The IGF1 gene was amplified in a final volume of 15 μL including 50-100 ng of genomic DNA, 0.375 μL of each primer (0.25 µM), 200 µM of dNTPs, 5 mM of MgCl2 and 1 U Taq polymerase. Primers used were those designed by Ge et al. (1997). The sequences of primers were IGF-F: 5'-ATTACAAAGCTGCCTGCCCC-3' and IGF-R: 5'-CACATCTGCTAATACACCTTACCCG-3' for forward and reverse primers respectively. Thermal cycle for performing PCR was accordance with Table 1. A step 2 to 4 was repeated 31 times.
Table 1 Thermal cycle for PCR
Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP)
In order to genotyping for all samples and PCR-RFLP analysis, the PCR products were digested in a total volume of 10 μl that included: 4 μL of PCR product, 4 μL of distilled water, 1 μL of enzyme buffer and 1 μL of HaeII enzyme incubated at 37 ˚C for 12 hour according to Yilmaz et al. (2005). Digested PCR products were mixed with 10x loading buffer and subjected to 2 percent agarose gel electrophoresis that contained Safe stain in 0.5x TBE. The genotyping of samples were done by comparison of digested PCR products and 50 bp DNA ladder.
Population genetic analysis was done using PopGene32. Effects with (P≤0.05) were considered significant. Associations were sought between IGF1 genotypes and lambs’ birth weight (BW), five-month weight (BW5) and average daily gain up to age five months (ADG). Least squared means (LSM) were used for multiple comparisons among genotypes using the general linear model (GLM) procedure in a fixed effect model in SAS (2004). Birth weight (BW,Yijk) was described thus:
Yijk= µ + Sexi + Tj + Gk + eijk
μ: overall LSM.
Sexi: effect of sex (i=1,2).
Tj: effect of the lambing year.
Gk: effect of IGF1 genotype (i=1,2,3).
eijk: residual random effect.
Five-month weight (BW5) and average daily gain up to age five months old (ADG) (Yijklm) were described thus:
Yijklm= µ + Sexi + b1BWj + b2Dimk + Tl + Gm + eijklm
b1: regression coefficient for BW on the dependent variable. BWj: effect of BW as a covariate.
b2: regression coefficient for the weaning period.
Dimk: effect of weaning period as a covariate.
Statistical interactions were not permitted between parameters.
RESULTS AND DISCUSSION
PCR-RFLP and population genetics
A 265 bp fragment was amplified from within exon 1 of ovine IGF1 (Figure 1). Digestion of that PCR product using HaeII revealed two alleles, hence three genotypes (Figure 2): AA (179, 86 bp), BB (265 bp) and AB (265, 179, 86 bp). All three genotypes were found in the present study, showing polymorphism. Alleles A and B were found with % frequencies 47.08 and 52.92. Genotypes AA, AB and BB were obtained with % frequencies 28.33, 37.50 and 34.17. Chi-squared and G-squared tests showed that the locus was not in Hardy-Weinberg equilibrium (P<0.01; Table 2).
IGF1 genotypes and early growth traits
No significantassociations were found between IGF1 genotypes and BW, BW5 or ADG (Table 3). However results approaching significance suggested that AA might have produced a 0.04 kg gain in BW and BB a 0.41 kg reduction in BW5. No effects on ADG were apparent. Detection of quantitative trait loci (QTL) has become a ‘hot’ topic since the late 1980s. Most studies have investigated the contribution of marker information into existing breeding programmes. As with most new technologies, it will probably be necessary to modify breeding programmes to exploit marker-assisted selection (Weller, 2009). Marker-assisted selection has so far been successful in commercial animals (Yilmaz et al. 2000). IGF1 has a remarkable diversity of biological effects on embryonic and postnatal growth.
Figure 1 PCR products using IGF-F and IGF-R primers analyzed by electrophoresis in a 1.5% agarose gel with safe stain
Figure 2 HaeII digestion of PCR products from animals those were genotyped.
Agarose gel (2%)
AA= 179 and 86 bp; AB= 265, 179 and 86 bp and BB= 265 bp
Table 2 Results of the χ2 and G2 test and probability levels in Hardy-Weinberg equilibrium
Table 3 Least square means and standard errors for early growth traits of Zandi sheep according to the different IGF1 genotypes
Circulating IGF1 concentrations correlate with fetal and neonatal size in several species. IGF1 promotes the growth of fetal organs, endocrine glands and skeletal maturation in fetal sheep, in part by enhancing fetal amino acid and glucose uptake. In postnatal life, IGF1 is a key determinant of animals’ linear growth, as a result of its effect on longitudinal bone growth (promoting osteoblast division and proliferation), muscle growth (enhancing myocyte differentiation and multiplication) and cartilage growth (increasing chondrocyte colony formation) (Honarvar et al. 2012). Mehmannavaz et al. (2010) found a significant effect of IGF1 polymorphism in Holstein bulls on estimated breeding values (EBV) for milk production traits. In Baluchi sheep Tahmoorespur et al. (2009) found a significant effect of IGF1 polymorphism in exon 1 on daily gain from birth to weaning but not for birth weight, weaning weight, weaning to six month and six month to yearling age. A study of Zel (tailed) and Lori-Bakhtiari (fat-tailed) sheep found no effect of polymorphisms in the 5’ flanking region of IGF1 with carcass traits (Honarvar et al. 2012). In Baluchi sheep Gholibeikifard et al. (2013) found no association between polymorphisms in exon 3 of IGF1 and birth weight, weaning weight, six-month weight, nine-month weight and yearling weight. Purbayramian et al. (2013) in Moghani sheep found no effect of IGF1 polymorphism on birth or yearling weight. The present study’s results hinted at genotypic effects on in early-growth traits. Allele A might have produced relatively high birth weight and five-month weight, however the differences were negligible and non significant. Those results might have arisen from small population size and the polygenicity of the traits. Investigation of particular alleles suggested that one allele, here called A, had a nearly-significant effect on birth weight and five month old weight. The genotypic distribution in the present study was not in Hardy-Weinberg equilibrium. Up to date little is known about molecular characteristics of Zandi sheep in Iran but migration and non random mating might contribute to the observed disequilibrium. In this study we used native breed sheep in a rural flock and we cannot expect that the suitable allele of IGF1 would have a significant effect. Variation in quantitative traits is controlled by multiple genes with limited effect and current results showed that IGF1 could not capture remarkable variation of early-growth traits in Zandi sheep. Therefore, study of sheep populations under selection programs would be useful.
The present study is the first research of IGF1 in Zandi sheep. The results presented here indicate that there are three genotypes in exon 1 of IGF1 in this population, but we found no significant association between this gene and early growth. However, we can confirm polymorphism of this gene in the population and it could be used as a tool in breeding programs. In some studies IGF1 polymorphism has significant association with growth traits in animals. Therefore, more research is recommended on other regions of IGF1 in larger populations.
The authors thank the Islamic Azad University, Karaj Branch for Research Laboratory Agriculture Faculty teamwork.