There is a need for further research to reveal the genetic potential of indigenous sheep breeds in Türkiye with regards to yield traits ¹¹. It is believed that selecting animals with the desired genotype for candidate genes related to yield traits will enhance the success of breeding programs ¹⁵. The improvement of genetic values in indigenous sheep breeds may be facilitated by selection studies utilizing known candidate genes with a relationship with growth, a polygenic trait ¹⁶. Hence, there is an increasing interest in identifying polymorphisms within potential candidate genes associated with crucial growth performance traits in the sheep genome and exploring their utilization options in selection ¹⁷.

The somatotropic axis, including key components such as the insulin-like growth factor 1 (IGF1) gene and the growth hormone (GH) gene, contains promising candidate genes related to meat production traits in livestock ¹⁸. IGF1 plays a significant role in various physiological and metabolic processes, including the enhancement of glycolysis uptake, increased lipid synthesis, stimulation of myogenesis, inhibition of apoptosis, and promotion of cell proliferation ¹⁹. Additionally, IGF1 is a significant gene for growth and development in both intrauterine and postnatal life ²⁰. The insulin-like growth factor 1 receptor (IGF1R), activated by IGF1, is a transmembrane receptor containing tyrosine kinase that regulates birth weight in addition to cell growth and differentiation ²¹. According to a study conducted by Louvi et al. ^22, it was shown that offspring mice with the silenced-IGF1R gene silenced had birth weights that were around 45% lower and experienced postnatal growth retardation. Studies conducted on various farm animals, such as cattle, buffalo, sheep, pigs, and chickens, have reported that the IGF1R gene is associated with growth performance traits such as periodic body weight and daily weight gain ^18,21,23-25. On the other hand, it has been proposed that the IGF1R gene plays a significant role in the function of granulosa cells in mice, hence its association with fertility in females ²⁶. Furthermore, in a study conducted by Yang et al. ^27, it was reported that the IGF1R gene is associated with superovulation in cattle.

The IGF1R gene, which includes 21 exons and 20 introns, is located on the 18th chromosome in sheep. The sheep IGF1R gene encodes a polypeptide containing 1367 amino acids, and it plays a crucial role in promoting cell proliferation and facilitating postnatal growth ^28,29. Ding et al. ³⁰ have reported the IGF1R gene as a candidate gene for improving early-stage live weights in sheep. Previous studies have shown that the g.195C>T polymorphism in the 12th intron of the IGF1R gene affects live weight gain in different sheep breeds ^23,31. This study was conducted to determine the polymorphism of IGF1R-RsaI (g.195C>T) located in the 12th intron and to compare the early live weight from birth to weaning age and birth type in the Akkaraman breed in Türkiye.

In the present study, we used 397 male Akkaraman breed lambs on four farms in the Kayseri province of Türkiye. These farms raise sheep with the same feeding, care, and environmental conditions. All lambs were also tagged with a small plastic ear tag after birth. All data, such as the weight and birth type of the lambs, were recorded according to the numbers on the ear tags.

Animals and DNA Extraction: The weight of the lambs was measured at birth, 30th, 60th, and 90th days. K3EDTA tubes containing an anticoagulant were used to take about 10 ml of blood from the jugular vein. Genomic DNA used in genetic analyses was isolated from total blood samples using the phenol-chloroform extraction method.

Molecular Genetic Analyses: A primer pair of the IGF1R gene (NCBI Accession number: KJ140106.1) recommended by Proskura and Szewczuk (23) was utilized in the PCR process (forward; 5’TCC CAA GTG GAG GTG AGT CT3’; reverse; 5’ATA AGC CAG CTT CCT GCA CAC3’). PCR amplification was carried out in total volume of samples 20 μL, containing 1.5 mM MgCl2, 200 μM each primer, 250 μM dNTPs, 10×PCR buffer, 1.5 U Taq DNA polymerase, 50 ng DNA template, and deionized water. The thermal cycling PCR conditions included initial denaturation at 94 °C/5 min, followed by 33 cycles consisting of denaturation at 94 °C/50 sec, annealing at 59.5 °C/1 min, extension at 72 °C/50 sec, and a last extension at 72 °C/5 min. The presence of PCR products with a length of 206 base pairs (bp) was confirmed by using 2% agarose gels. The obtained PCR products were digested by incubating at 37°C for at least 5 hours with 5 U of the RsaI restriction endonuclease enzyme (Thermo Scientific). Following the digestion process, the DNA restriction fragments were separated in 2% agarose gels. The results of gel electrophoresis separations were visualized under UV light.

Statistical Analyses: The allele and genotype frequencies of the population in terms of IGF1R gene polymorphism were calculated manually. Chi-Square (χ²) test was used to identify the Hardy-Weinberg equilibrium of the population. The statistical significance of the relationship between lamb birth type (single or twin) and genotypes (AA, AB, BB) was tested using the Chi-Square analysis. In this study, the effects of IGF1R genotypes on the live weight of Akkaraman lambs were evaluated using the Generalized Linear Model (GLM). The model incorporates genotypes and other control variables to assess their impact on live weight. The mathematical representation of the model is as follows:

The β coefficients represent the effects of independent variables on live weight, estimated through GLM analysis. Each coefficient reflects the average effect of the respective variable on live weight.

Additionally, Pearson correlation coefficients were calculated to assess the relationship between genotypes and live weights at different growth periods (birth, 30th day, 60th day, and 90th day). The correlation values between genotypes (AA, AB, BB) and live weights, along with other calculations, were generated using JMP software (SAS Institute Inc., Cary, NC). These correlations provide important insights into how different genotypes influence growth rates at various stages of lamb development.

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Figure 1: Electrophoretic pattern of the PCR amplicons, which are 206 base pairs in length in the IGF1R gene, M: 100 bp DNA Ladder

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Figure 2: Electrophoretic pattern of RFLP fragments for IGF1R-RsaI polymorphism, AA: 206 bp, BB: 139, 67 bp, AB: 206, 139, 67 bp, M: 100 bp DNA Ladder

The analysis revealed that the AB genotype (0.49) was the most prevalent genotype, while the AA genotype (0.20) was the least common genotype. The analyses of the population indicated that the B allele (0.56) exhibited the highest prevalence among alleles, as shown in Table 1. Akkaraman lambs were found not to be in Hardy-Weinberg equi

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Table 1: Allele and genotype frequencies of IGF1R-RsaI polymorphism in Akkaraman lambs

Correlations of genotypes according to live weights at different growth times are given in Table 2. When analyzing the relationship between birth type (single or twin) and IGF1R genotypes, distinct effects of different genotypes on birth weight and subsequent live weights were observed. The study identified and statistically supported differences in weight gain based on the AA, AB, and BB genotypes of lambs, alongside their birth types. For single-born lambs, weight differences based on genotypes were noted. The average birth weights for AA, AB, and BB genotypes were recorded as 4557 g, 4617 g, and 4644 g, respectively. For twin-born lambs, AA genotype lambs weighed 4750 g, while AB genotype lambs weighed 4621 g at birth. Despite these differences, the variations in birth weight were not statistically significant (p>0.05). At 30 days, the average weight of single-born AA genotype lambs was 12.503 g, while AB and BB genotype lambs had average weights of 12.505 g and 12.156 g, respectively. For twin-born lambs, AA genotype lambs reached an average weight of 12.972 g, and AB genotype lambs weighed 13.170 g. However, the differences in 30-day weights between genotypes were also not statistically significant (p=0.5398). By day 60, the average weight of single-born AA genotype lambs was 22.281 g, while AB genotype lambs weighed 21.591 g, and BB genotype lambs reached 21.004 g. For twin-born lambs, AA genotype lambs averaged 22.523 g, while AB genotype lambs weighed 22.344 g. As with previous periods, the differences in 60-day weights were not statistically significant (p>0.05). At 90 days, single-born AA genotype lambs reached an average weight of 33.023 g, AB genotype lambs 31.809 g, and BB genotype lambs 31.637 g. For twin-born lambs, AA genotype lambs weighed 33.909 g, and AB genotype lambs reached 31.500 g. Again, there were no statistically significant differences between the 90-day weights of different genotypes (p>0.05).

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Table 2: Correlations of genotypes according to live weights at different growth times

A GLM analysis was performed to analyze the impact on birth weight and the weights at the 30th, 60th, and 90th days based on the data reported in Table 3. All models were computed via the highest likelihood technique. The farm factor exerted the most substantial influence on birth weight (LogWorth=19.047, p<0.0001). Furthermore, the weight at 30 days markedly affected the delivery weight (LogWorth=2.250, p=0.0056). The effects of the allele, 60th day weight, birth type, and 90th day weight were not statistically significant. The model was substantially affected by the farm variable (p<0.0001), but the allele variable exhibited questionable significance. The parameter estimates revealed that the AB allele exerted a favorable influence (estimate=108.133, p=0.0472), whereas the AA allele was not statistically significant (estimate=-86.806, p=0.2740). The farm component was found to be significant for the weight on the 30th day (LogWorth=22.602, p<0.0001). Alleles, birth type, and 90th-day weight were not significant in the model; however, birth weight was significant. The parameter estimates revealed that the weight on the 30th day was strongly influenced by farm 1 (estimate=-1004.637, p<0.0001), whereas farm 3 exerted a considerable positive effect (estimate=1199.090, p<0.0001). The AA allele was not significant, whereas the AB allele (estimate=-162.642, p=0.0882) exhibited marginal significance. For the weight at day 60, the weight at day 30 was the most important variable (LogWorth=38.125, p<0.0001), and the farm factor was also noteworthy (LogWorth=22.602, p<0.0001). Birth weight, allele, and birth type were found to be statistically insignificant. The AB allele had a positive and significant influence on parameter estimates (estimate=218.564, p=0.0309), while the AA allele was not significant (estimate=11.042, p=0.9404). The farm component exerted a substantial influence (LogWorth=9.260, p<0.0001), with the weight on the 60th day identified as the most important variable affecting the weight on the 90th day (LogWorth=55.143, p<0.0001). The model indicated that birth type (LogWorth=0.856, p=0.1394), allele (LogWorth=0.697, p=0.2010), and 30th-day weight (LogWorth=0.107, p=0.7818) were not statistically significant. According to parameter estimates, Farm 1 exhibited a positive and substantial influence (estimate=1244.934, p<0.0001), whereas Farm 3 demonstrated a negative and significant impact (estimate=-1343.843, p<0.0001). The AB allele showed a marginally significant negative impact (estimate=-297.512, p=0.0755).

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Table 3: Generalized Linear Model (GLM) Results for the Effect of IGF1R Farm, Genotypes and Birth Weight on the Live Weights of Akkaraman Lambs

The study conducted on the Akkaraman population revealed a higher prevalence of the BB genotype among lambs with a single birth type, as indicated in Table 4.

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Table 4: Distribution of IGF1R genotypes among birth types of Akkaraman lambs

The effects of polymorphisms in the IGF1R gene on growth traits have also been investigated in studies conducted on different livestock species. A study investigating Hereford cattle breeds reported that IGF1R variants were significantly associated with body weight and body measurement traits, while another study investigating Angus cattle breeds reported a significant association with weaning weight ^18,32. The IGF1R gene variant has been linked to the body weight and measurement traits of yaks in China ³³. A significant IGF1R variant has been reported in Egyptian buffaloes, associated with high daily live weight gain from birth to 6 months ²⁴. The investigated IGF1R variant in Wanbai and Yorkshire pig breeds has been reported to be associated with higher body weight at birth, 2 months, and 6 months ²⁵. According to a study conducted on chickens, it was found that the IGF1R gene has a significant effect on early growth traits ³⁴. A study conducted on Japanese quails reported that the IGF1R gene is significantly associated with 10-week live weight and average live weight gains at different periods ³⁵. Studies conducted on sheep have also reported associations between the IGF1R gene and growth traits. In Makooei sheep, the IGF1R gene has been reported to be associated with average daily weight gain from birth to 6 months ³⁶. A study on a Merino-derived sheep breed in Poland investigated the SNP (c.654G>A) in the 3rd exon of IGF1R, revealing an association between the gene and average weight gain from 56–78 days after birth ²⁹. Another study conducted on Hulun Buir sheep reported a significant association between IGF1R and various growth traits ³⁰. In a study of Pomeranian Coarsewool sheep breed in Poland, the investigated SNP (g.195C>T) was reported to be significantly associated with body weight on days 1, 33, and 90 post-birth, as well as with average daily weight gain between days 1-33, 33-90, and 1-90 ²³. Similarly, Karadağ ³¹ reported that in Alman Karabaş × Kıvırcık, Hampshire Down × Merino, Kıvırcık, Karacabey Merino, and Ramlıç breeds in Türkiye, lambs carrying the AB genotype have higher birth weights. In this study, the IGF1R genotype was found to have no statistically significant effect on lamb weights across all measured periods (birth, 30th, 60th, and 90th days). However, birth type (single or twin) and farm conditions were identified as significant factors affecting weight gain in all measurement periods. These findings highlight the need to consider both genetic and environmental factors when evaluating the growth performance of lambs.

Yang et al. ²⁷ established a relationship between the IGF1R gene and superovulation in cattle. The literature review shows that in addition to studies on the effects of the IGF1 gene on the twinning trait, there is also a need for studies on IGF1R, the receptor gene of IGF1. In addition to growth, the twinning rate is a crucial economic trait that could be utilized for sheep breeding objectives. In this study investigating the IGF1R-RsaI polymorphism in sheep, it was observed that the BB genotype was more prevalent in single-birth lambs. The findings of this study suggest that future studies investigating the relationship between IGF1R-RsaI polymorphism and twinning are necessary. In summary, this study found that IGF1R-RsaI polymorphism was effective on live weight in lambs according to birth type. Future research may investigate the IGF1R gene for more growth traits or use a larger population of Akkaraman sheep breeds. Additional to these findings, the fact that the BB genotype was more prevalent in single-birth lambs might prove an important area for future research for the purpose of establishing a possible link with lambing. These findings are a foundational investigation of genetic factors that affect growth and lambing traits in Akkaraman sheep, highlighting the complexity of genetic effects on these traits and the necessity for more comprehensive studies.

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