Growth and carcass traits associated with GH1/Alu I and POU1F1/Hinf I gene polymorphisms in Zebu and crossbred beef cattle

RESEARCH ARTICLE

Growth and carcass traits associated with GH1/Alu I and POU1F1/Hinf I gene polymorphisms in Zebu and crossbred beef cattle

Rogério A. Curi^I; Darío A. Palmieri^I; Liliane Suguisawa^II; Henrique N. de Oliveira^II; Antonio C. Silveira^II; Catalina R. Lopes^I

^IUniversidade Estadual Paulista 'Júlio de Mesquita Filho', Instituto de Biociências, Departamento de Genética, Botucatu, SP, Brasil

^IIUniversidade Estadual Paulista 'Júlio de Mesquita Filho', Faculdade de Medicina Veterinária e Zootecnia, Departamento de Melhoramento Zootécnico e Nutrição Animal, Botucatu, SP, Brasil

^{Send Correspondence to}

ABSTRACT

The objectives of the present study were to estimate the allele and genotype frequencies of the GH1/Alu I and POU1F1/Hinf I polymorphisms in beef cattle belonging to different genetic groups and to determine the effects of these polymorphisms on growth and carcass traits in cattle submitted to feedlot management, an intensive production model. Genotyping was performed on 384 animals, including 79 Nellore, 30 Canchim (5/8 Charolais + 3/8 Zebu), 30 Simmental x Nellore crossbred and 245 Angus x Nellore crossbred cattle. Body weight, weight gain, dressing percentage, Longissimus dorsi area and backfat thickness were fitted using the General Linear Model (GLM) procedure of the SAS program and the least square means of the genotypes were compared using the F test. The results showed significant associations between the LL genotype of the GH1/Alu I polymorphism and higher weight gain and body weight at slaughter (p < 0.05). The POU1F1/Hinf I polymorphism did not have any effect on the growth and carcass traits analyzed.

Key words: beef cattle, polymorphisms, candidate gene, growth, carcass.

Introduction

Most traits of economic interest are of a quantitative nature and are controlled by a large number of genes which each contribute a small effect to the trait, the loci responsible for such traits being known as quantitative trait loci (QTL). However, there is debate regarding the existence of principal genes which contribute more to the phenotypic variation of a quantitative trait. Weight gain, feed efficiency, meat tenderness, marbling fat and backfat are some of the main quantitative traits evaluated in beef cattle bulls, although these analyses have been performed with little knowledge about the genetic and biochemical bases of these traits.

During the various growth phases of ruminants with different frame sizes differences in metabolism have been observed, somatotrophic axis hormones being a very important factor in such differences (Owens et al., 1993). The somatotrophic axis play a key role in the regulation of the metabolism and physiology of mammalian growth and essentially consists of growth hormone (GH), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) and their associated binding proteins (BP) and receptors (R), i.e. IGFBP, GHR, IGF-1R and IGF-2R.

The GH1 gene codifies for circulating growth hormone which binds to the extracellular domain of specific transmembrane growth hormone receptors (GHR) which are expressed in various tissues, especially liver-tissue. This process is the signal for a cascade of intracellular metabolic events culminating in the production of IGF-1 by the target tissues, of which liver-tissue is the most important (Renaville et al., 2002).

Directly or indirectly, through the action of IGF-1, growth hormone is the main regulator of postnatal somatic growth, stimulating anabolic processes such as cell division, skeletal growth and protein synthesis. In addition, growth hormone is involved in the regulation of fat oxidation (lipolytic activity), inhibition of glucose transport to peripheral tissues (diabetogenic activity) and the regulation of the activity of ribosomes involved in the translation, which in turn influences protein synthesis (Goodman, 1993). These processes are directly implicated in the metabolism of nutrient distribution to different tissues and, consequently, in carcass composition and quality (Schlee et al., 1994a). The growth hormone factor 1 gene (POU1F1) can be considered a candidate gene for growth and carcass traits in cattle since its product, a pituitary-specific positive transcription factor 1, regulates the transcription of GH1 and the prolactin gene in the mammalian anterior pituitary (Bodner et al., 1988). Pituitary-specific positive transcription factor 1 was subsequently characterized as an activator of other pituitary genes, including POU1F1 itself (Chen et al., 1990) and the growth hormone releasing hormone gene (GHRH), among others (Lin et al., 1992).

Lucy et al. (1991) described a C ® G exchange at codon 127 in the bovine GH1 gene (GH1/Alu I polymorphism) leading to a leucine (L allele) to valine (V allele) substitution which have been associated with several production traits in cattle such as growth and carcass composition and quality (Schlee et al., 1994a, b; Regitano et al., 1999; Sartore and Di Stasio, 2000). Moody et al. (1995) identified in bovines the A and B alleles of the POU1F1/Hinf I polymorphism, which is an exon 6 A ® G silent mutation of the bovine POU1F1 gene (Dierkes et al., 1998) which probably is not the direct responsible by phenotypic variation, although relationships have been observed between POU1F1 polymorphisms and body weight and somatic measures (Renaville et al., 1997), weaning and yearling weight (Carrijo et al., 2003).

The objectives of the present study were to estimate the allele and genotype frequencies of the different alleles of the GH1/Alu I and POU1F1/Hinf I polymorphisms of beef cattle belonging to different genetic groups and to determine associations between these polymorphisms and the performance of the animals when submitted to an intensive production model.

Material and Methods

Cattle

The study was carried out in the experimental feedlot facility of the Animal Science Department of São Paulo State University (Unesp, Botucatu-SP, Brazil) during three consecutive years (2000 = feedlot 1, 2001 = feedlot 2 and 2002 = feedlot 3) using samples of cattle from commercial herds belonging to six different farms. The sample group consisted of 384 bull calves belonging to four different genetic groups differing in frame size, these groups being Bos indicus (Zebu) Nellore pure-bred cattle (n = 79) and Bos taurus (European)/Zebu cross-breeds consisting of Canchim cattle (5/8 Charolais + 3/8 Zebu, n = 30) and 1/2 Simmental (n = 30) and 1/2 Angus (n = 245) cross-breeds resulting from crosses between Simmental or Angus sires and Nellore dams.

The bull calves were weaned at 210 days using a creep-feeding system. At the beginning of the experiment the bulls were individually identified, treated against endo- and ectoparasites, divided into groups of five animals in each feedlot pen according to breed , where they were fed diets formulated according to the norms of the National Research Council (NRC, 1996) for an average daily gain in excess of 1.2 kg. After entering the pen, the bulls were allowed to adapt for about 20 days. When they were slightly over one year old, and after an average feedlot (fattening) period of 110 days, the bulls were slaughtered at a commercial abattoir.

For growth traits the bulls were weighed to assess their body weight (BW) when first penned (BW₀) and also at the beginning of their feedlot period (BW₁) and close to the time of slaughter (BW_END). Average daily weight gain (ADWG) was calculated for the interval between the last two weight measurements. For carcass traits, ribeye area (REA) or Longissimus dorsi area and backfat thickness (BT) were assessed at BW_END by ultrasonography using the method of Perkins as modified by Gresham (1998) and carcass weight (CW) was measured at slaughter with dressing percentage (DP) being taken as CW divided by BW_END.

Extraction of DNA and genotyping

For each bull, 5 mL of total blood was collected from the left jugular vein using vacuum tubes containing 7.5 mg of EDTA and genomic DNA extracted from a 300-µL aliquot using the Genomic Prep^TM Blood DNA Isolation kit according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, USA).

Bulls were genotyped for the GH1 and POU1F1 genes by using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). The chromosome 19 GH1/Alu I forward primer was 5'-GCT GCT CCT GAG GGC CCT-3' and the reverse primer 5'-GCG GCG GCA CTT CAT GAC CCT-3' and the annealing temperature 58 °C while the chromosome 1 POU1F1/Hinf I forward primer was 5'-CAA TGA GAA AGT TGG TGC-3' and the reverse primer 5'-TCT GCA TTC GAG ATG CTC -3'with an annealing temperature of 54 °C.

To determine the GH1 gene L and V alleles we amplified a 223-bp fragment located between intron 4 and exon 5 and digested it with the Alu I restriction enzyme (Schlee et al., 1994a). The POU1F1 gene A and B alleles were identified by amplification of a 1301-bp sequence corresponding to fragments of exons 5 and 6 followed by digestion with Hinf I (Moody et al., 1995).

Each PCR was performed in 25 µL of amplification mixture consisting of 50 ng genomic DNA, 0.20 µM of each primer, 10 mM Tris-HCl, pH 8.0, 50 mM KCl, 2.0 mM MgCl₂, 0.2 mM of each dNTP and 1 unit of Taq DNA polymerase. The amplification protocol was initial double- strand denaturation at 94 °C for 4 min, followed by denaturation at 94 °C for 1 min, primer annealing at 54 to 58 °C for 45 s (depending on the primer), extension at 72 °C for 1 min, a final extension at 72 °C for 4 min. Steps 2, 3 and 4, corresponding to one cycle, were repeated 35 times. The amplified fragments were digested in a reaction mixture containing 10 µL of PCR product and 3 units of the restriction enzyme and the digested fragments separated by 3% (w/v) agarose gel horizontal electrophoresis using a 100 base pair (bp) molecular weight standard (Invitrogen, São Paulo, SP, Brazil) to calculate the size of the amplified and digested fragments which were visualized by ethidium bromide staining and exposure to ultraviolet light. The genotypes of the individual cattle were determined for each polymorphism by analyzing the size of the fragments reported as base pairs.

Statistical analysis

Genotype and allele frequencies were calculated for each polymorphism according to Weir (1996). Differences in the allele frequencies of the polymorphisms within and between genetic groups were determined by the method of Goodman adapted by Curi and Moraes (1981).

The traits of interest were analyzed by least square analysis of variance (p = 0.05) using the General Linear Model (GLM) procedure of the SAS program (Statistical Analysis System, 1999). The linear model used to fit the quantitative variables included, in addition to the genotype effect, the contemporary group effect which considered the interaction between genetic groups (1, ..., 4), feedlots (1, ..., 3) and farms of origin (1, ..., 6) as follows: Y_ijk = µ + G_i + GC_j + e_ijk, where Y_ijk = production trait, µ = overall mean, G_i = fixed effect of the i^th genotype, GC_j = fixed effect of the j^th contemporary group, and e_ijk = random error.

Genotypes with very low frequency (less than 0.10) in the total sample of bulls or genetic groups showing a single genotype were not included in the analysis in order to avoid unreliable results or confounding the influence of genetic groups and genotype effects on traits of interest. The sire effect was not included in the linear model since the number of genotyped bulls which were progenies of the same sire was very small (8.5 on average). The possibility of confounding the influence of genotype effect and sire effect on production traits was low because of the large number of small half-sib families.

Results

We detected the L and V alleles of the GH1/Alu I polymorphism in our sample of bulls, but although the LL and LV genotypes were present the VV genotype was not (Figure 1). The A and B alleles of the POU1F1/Hinf I polymorphism were present in our sample as the AA, AB and BB genotypes (Figure 2). The GH1/Alu I and POU1F1/Hinf I polymorphism allele frequencies are shown in Table 1 and the genotype frequencies in Table 2.

The GH1/Alu I polymorphism showed segregation in the Canchim, 1/2 Simmental and 1/2 Angus genetic groups. The GH1/Alu I polymorphism L allele was fixed in Nellore bulls, resulting in the occurrence of the LL genotype only, and its frequency was significantly higher than that of the V allele in all the other genetic groups studied. Nellore animals presented a significantly higher frequency of the L allele than the Canchim and 1/2 Angus groups, which in turn showed higher frequencies than the 1/2 Simmental group. The LL genotype predominated in Canchim and 1/2 Angus bulls, while a higher frequency of the LV genotype occurred in the 1/2 Simmental group.

The POU1F1/Hinf I polymorphism segregated in all four genetic groups, with the A allele showing a significantly higher frequency than the B allele in all groups. The frequency of the A allele was significantly higher in the Nellore, Canchim and 1/2 Simmental groups compared to the 1/2 Angus group. We did not detect the POU1F1/Hinf I polymorphism BB genotype in either the Nellore or 1/2 Simmental bulls and its frequency was very low in the other two groups.

The least square means and standard errors of quantitative growth traits for the different genotypes of the GH1/Alu I and POU1F1/Hinf I polymorphisms are shown in Table 3, the same parameters for carcass traits being shown in Table 4.

A significant effect of the GH1/Alu I polymorphism genotypes was observed on BW_END (p = 0.0241), average daily weight gain (p = 0.0220) and carcass weight (p = 0.0441), with the homozygous LL genotype being favorable compared to the heterozygous LV genotype. No significant effect was observed on BW₀ (p = 0.8982), BW₁ (p = 0.8704), dressing percentage (p = 0.3496), ribeye area (p = 0.8723) or backfat thickness (p = 0.5898).

Only the AA and AB genotypes were considered in the comparison analysis between the POU1F1/Hinf I polymorphism genotypes and production traits. The results showed no significant effect of genotypes on the growth traits BW₀ (p = 0.2265), BW₁ (p = 0.1353), BW_END (p = 0.4437) and average daily weight gain (p = 0.6831) or the carcass traits carcass weight (p = 0.2790), DP (p = 0.8165), ribeye area (p = 0.7855) and backfat thickness (p = 0.9514).

Discussion

The GH1/Alu I polymorphism identified by Lucy et al. (1991) consists of a cytosine to guanine exchange at codon 127 in the GH1 gene and the substitution of the amino acid leucine (L allele) by valine (V allele) to produce different forms of the growth hormone which can differ in receptor-binding efficiency and thus modify physiologic processes and produce different phenotypes.

In our study, the L allele frequencies obtained were similar to results reported in the literature which showed a fixation of this allele in Zebu breeds (Kemenes et al., 1999) and its predominance in the taurine breeds Charolais (Kemenes et al., 1999), Piedmontese (Di Stasio et al., 2002), Simmental and Angus (Vasconcellos et al., 2003), with frequencies of 0.72, 0.72, 0.82 and 0.77, respectively. Silveira (2002) obtained a frequency of 0.91 for the L allele in the synthetic Canchim breed.

We found significantly higher weight gain and body weight at slaughter in LL animals compared to LV animals. Because the VV genotype was absent from our sample the type of allele interaction could not be determined. Other authors have shown that GH1/Alu I polymorphism can influence performance traits in beef cattle, with Schlee et al. (1994b) having shown that in Simmental cattle the LV genotype is associated with higher weight gain while cattle with the VV genotype presented better carcass scores. Regitano et al. (1999) observed a significant linear increase in the V allele over four generations in a Canchim herd and suggested that this polymorphism may be associated with phenotypic traits that are subject to selection in this herd. Sartore and Di Stasio (2000) also suggested the occurrence of indirect selection for the V allele in Piedmontese cattle, although in a subsequent study (Di Stasio et al., 2002) these authors found no evidence of an association between this polymorphism and growth or carcass traits in this breed. Tambasco et al. (2003) observed that LL genotype Canchim x Nellore, Simmental x Nellore and Angus x Nellore crossbred cattle presented higher weight gain from birth to weaning as compared to the LV genotype, although from weaning to yearling the opposite was true. In a review on the molecular genetics of beef cattle, Switonski (2002) states that most studies have shown that cattle with the VV genotype show lower growth rates as compared to the other two genotypes. Due to these contradictory results, Switonski (2002) concluded that the use of the GH1/Alu I polymorphism in beef cattle selection programs is still premature.

The A and B alleles of the POU1F1/Hinf I polymorphism were identified by Moody et al. (1995). This polymorphism is a silent mutation (A ® G) located in exon 6 of the bovine POU1F1 gene (Dierkes et al., 1998) and, therefore, probably does not cause any phenotypic variations.

The frequencies reported in the literature for the A allele of the POU1F1/Hinf I polymorphism were 0.25 for Piedmontese animals (Di Stasio et al., 2002), 0.53 for Belgian Blue cattle (Renaville et al., 1997), 0.45 for Angus cattle (Moody et al., 1995), and 0.86 for the synthetic Canchim breed (Carrijo et al., 2003). We did not find allele frequencies of this polymorphism for pure Zebu breeds.

Our results for the POU1F1/Hinf I polymorphism showed no significant effect of the on growth or carcass traits, indicating that this is not a molecular marker associated with meat production in beef cattle and supporting the work of other published studies (Zwierzchowski et al., 2001; Di Stasio et al., 2002; Zhao et al., 2004). However, Renaville et al. (1997) reported that in taurine Belgian Blue cattle, POU1F1/Hinf I BB animals were significantly superior to animals with the AB and AA genotypes in terms of body weight at 7 months of age and Carrijo et al. (2003) found that for the mixed race taurine/zebu Canchim breed the BB genotype was superior to the AB and AA genotypes for weaning and yearling weight.

The differing results reported in the literature suggest that the GH1/Alu I and POU1F1/Hinf I polymorphisms are not directly responsible for phenotypic variations and these contradictions can be explained by differences in the linkage disequilibrium between markers and quantitative trait loci (QTL) in the various populations studied, by different epistatic interactions between the genetic bases of these populations and QTL, or even by the experimental design and statistical approach followed. However, the fact that our results for the GH1/Alu I polymorphism showed that the LL genotype was superiority to the LV genotype for growth and carcass traits justifies continuing studies on this polymorphism with Zebu and crossbred cattle.

Acknowledgments

The authors thank Universidade Estadual Paulista (Unesp) for providing the infrastructure for this study and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financing the study.

Received: December 2, 2004; Accepted: July 5, 2005.

Associate Editor: Pedro Franklin Barbosa

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Publication Dates

• Publication in this collection
  10 Mar 2006
• Date of issue
  2006

History

• Received
  02 Dec 2004
• Accepted
  05 July 2005