Association of GH polymorphisms with growth traits in buffaloes

Highlights

• Two missense mutations were detected in exon5: a p.Leu153Val SNP and a novel p.Asn174His SNP.

• At weaning age, animals with the Leu allele had higher weaning body weight and gain.

• Animals with Leu/Asn and Leu/His haplotypes had higher GH serum, mRNA, and protein levels at weaning.

• At yearling age, animals with Val and His alleles had higher yearling weight and gain.

• Animals with the Val/Asn haplotype had increased GH, and IGF1 serum, mRNA, and protein and GHR mRNA and protein levels.

Abstract

Members of the somatotrophic axis, especially GH and IGF1, are essential for growth. The association between GH polymorphisms and growth traits was numerously studied in cattle; however, no data are available for such association studies in buffalo. Therefore, this study was conducted to screen for polymorphisms in the GH gene and to study their putative association with growth traits in 200 Egyptian buffaloes. Polymerase chain reaction single-strand conformation polymorphism and sequencing were applied to look for polymorphisms in 3 loci spanning all exons and introns of buffalo GH. The C (MspI+) >T (MspI-) SNP in intron3, which is well known in cattle, was not detected in the examined buffaloes. However, 2 missense mutations were detected in exon5: one previously detected p.Leu153Val SNP, with very low frequencies for the mutant (Val) allele and one novel p.Asn174His SNP. At weaning age, the p.Leu153Val SNP was significantly associated with weaning body weight and gain with the positive effect of the wild allele (Leu) and higher GH serum, mRNA, and protein levels in animals with Leu/Asn and Leu/His haplotypes. At yearling age, the 2 SNPs associated with yearling weight and gain with positive effect for the mutant (Val and His) alleles with increased GH, and IGF1 serum, mRNA, and protein and GHR mRNA and protein levels in animals with Val/Asn haplotype. Therefore, the selection of Egyptian buffaloes with the Val/Asn haplotype could improve the growth traits of Egyptian buffaloes at yearling age which is the target age for perfect growing.

Introduction

In Egypt, water buffaloes (Bubalus bubalis) are always reared for a dual purpose of milk and meat production. Rearing of buffaloes has many advantages as compared with cattle. Buffaloes can easily adapt to different harsh environmental conditions, can efficiently use low quality and less digestible feeds, and can resist diseases better than cattle [[1], [2], [3]]. In accordance with the last estimate in 2011, over 41% of Egypt's total red meat production is from buffaloes' beef and veal [4]. Buffalo carcass weights are greater than cattle [5], and their meat is healthier (less saturated fat and cholesterol, but more protein and mineral) and tastier than beef [6]. Despite these advantages, Egyptian buffaloes remain underused and show suboptimal meat production potential. Thus, genetic improvement through the application of marker-assisted selection (MAS) for superior production traits ranks high among our agricultural research needs. Detection and validation of genetic markers associated with growth traits are essential steps in MAS system.

Growth traits in bovine are mainly regulated by somatotrophic axis–related genes and proteins such as GH, growth hormone receptor (GHR), and IGF-1 [7,8]. The GH gene (which assigned to chromosome 19 and consists of 5 exons and 4 introns) is considered as a positional and functional candidate gene for traits with economic values such as growth, carcass, and milk traits in ruminants [[9], [10], [11], [12], [13], [14], [15], [16]]. This gene encodes GH protein, which is an anabolic hormone synthesized by the somatotrophic cells of the anterior pituitary [17]. As a leading member of the somatotrophic axis, GH plays a crucial role in growth, reproduction, and lactation mainly through induction of cell proliferation, protein and lipid formation and metabolism [10,14,18]. Growth hormone actions mediated through binding to its receptor, GHR, which is expressed in several tissues, particularly the liver, muscles, and adipose tissues [19]. This specific binding activates the JAK2/STAT5 signaling pathway, which in turn induces expression of downstream target genes such as IGF1 [20]. Bones' and muscles' growth-promoting effects of GH are mediated by GHR and IGF1 [21]. Therefore, identifying polymorphisms in somatotrophic axis genes (GH, GHR, IGF1) is very crucial for the successful MAS program of growth traits [22].

Associations between GH polymorphisms and growth and milk traits are mostly performed on cattle and some in goats and sheep, but not in buffalo so far. Among numerous polymorphisms detected in bovine GH, 2 SNPs have been extensively studied for their significant role in different economic traits: a C (MspI⁺) >T (MspI⁻) SNP in intron3 and the missense mutation C (Leu, AluI⁺) >G (Val, AlI⁻) in exon5 (E5). Associations of these 2 SNPs with milk traits were greatly studied, and the effects were well distinct and had no contradictions. Most studies concluded that cattle with the wild alleles (Leu) had higher milk and protein yield, but the lower fat % than the heterozygous (Leu/Val) animals [[23], [24], [25], [26], [27]]. Subsequently, the presence of the mutant allele (Val) had a lower milk yield with nearly 240 kg decrease [28]. Unlike milk traits, there were controversial data regarding the effect of these 2 SNPs on growth and carcass traits. Some studies reported significant effects for a C (MspI⁺) >T (MspI⁻) SNP on growth traits [29], but other studies showed no effect [30,31]. Similarly, some studies favored the Val allele for a positive effect on growth and carcass traits [7,11,32], but other studies either favored positive effect for the Leu allele [13,24,32] or no effect for either [31,33].

A nonsynonymous C > T (p.Thr198 Met) SNP in E5 was associated with growth and carcass traits, with the T allele showing increased weaning body weight (WW), higher carcass marbling and weight [13,34]. In addition, a synonymous c. 607A > C (p. Arg203) SNP in E5 was also associated with WW and some carcass traits, with superior effect for the C allele [11,13,24]. Some other SNPs in the GH promoter and 3′UTR were also associated with body conformation and depth and carcass fat [24]. On the other hand, the effect of a C > T (p.Thr200Met) SNP in E5 was not identified for growth or carcass traits [35].

In buffalo GH, to date, studies only focus on the detection of polymorphisms. The C (MspI⁺) >T (MspI⁻) SNP in intron3 was identified in Indian buffaloes [36]. The C (Leu, AluI⁺)>G (Val, AluI⁻) SNP in E5 was detected in Egyptian [37], Indian [36], Indonesian [38], Iraqi [39], and Iranian [40] water buffaloes with very low frequency or absence of the mutant (Val) allele. Besides, Indian buffaloes only fixed the C allele of the silent mutation c.607 A > C(p.Arg203) in E5 [36]. In Iranian water buffaloes, Eghbalsaied et al [40] found 3 other SNPs in E5 of GH: G > A (p.Lys165), C > A (p.Glu166Lys), and G > A (p. Glu166). None of these previous researches studied the associations between these GH SNPs and growth traits in buffaloes. In addition, these studies focused only on small fragments of GH; however, to date, no whole GH screening for polymorphisms was performed in buffalo. No enough data are available in publications regarding functional validation of GH polymorphisms associated with growth traits in ruminants. Subsequently, the effect of these polymorphisms on gene/protein expression and hormonal levels has not been elucidated yet.

This study is a part of a broader project to improve the genetic potentiality of Egyptian water buffalo. We previously found novel SNPs in IGF1, IGF1R, IGF2, IGF2R, GHR, and Cyp19A1 and explored their associations with growth traits, milk performance, and fertility [[41], [42], [43], [44], [45], [46]]. Thus, as part of the MAS program targeting the enhancement of Egyptian buffalo growth traits, we conducted this study to screen most of the GH gene (all 5 exons and 4 introns) for polymorphisms, to study their associations with growth traits, and to validate this effect through detection of the associated molecular and hormonal changes.