Genetic Variations of MSTN and Callipyge in Tibetan Sheep: Implications for Early Growth Traits.

Tibetan sheep are vital to the ecosystem and livelihood of the Tibetan Plateau; however, traditional breeding methods limit their production and growth. Modern molecular breeding techniques are required to improve these traits. This study identified a single nucleotide polymorphism (SNP) in myostatin (MSTN) and Callipyge in Tibetan sheep. The findings indicated notable associations between MSTN genotypes and growth traits including birth weight (BW), body length (BL), chest width (ChW), and chest circumference (ChC), as well as a particularly strong association with cannon circumference (CaC) at 2 months of age. Conversely, Callipyge polymorphisms did not have a significant impact on Tibetan sheep. Moreover, the analyses revealed a significant association between sex and BW or hip width (HW) at 2 months of age and ChW, ChC, and CaC at 4 months of age. Furthermore, the study's results suggested that the genotype of MSTN as a GA was associated with a notable sex effect on BW, while the genotype of Callipyge (CC) showed a significant impact of sex on CaC at 2 months of age. These results indicated that the SNP of MSTN could potentially serve as a molecular marker for early growth traits in Tibetan sheep.

Livestock species as emerging models for genomic imprinting.

Genomic imprinting is an epigenetically-regulated process of central importance in mammalian development and evolution. It involves multiple levels of regulation, with spatio-temporal heterogeneity, leading to the context-dependent and parent-of-origin specific expression of a small fraction of the genome. Genomic imprinting studies have therefore been essential to increase basic knowledge in functional genomics, evolution biology and developmental biology, as well as with regard to potential clinical and agrigenomic perspectives. Here we offer an overview on the contribution of livestock research, which features attractive resources in several respects, for better understanding genomic imprinting and its functional impacts. Given the related broad implications and complexity, we promote the use of such resources for studying genomic imprinting in a holistic and integrative view. We hope this mini-review will draw attention to the relevance of livestock genomic imprinting studies and stimulate research in this area.

Proteomic and metabolomic profiling reveals the involvement of apoptosis in meat quality characteristics of ovine M. longissimus from different callipyge genotypes.

Proteome and metabolome changes in muscles from callipyge mutation (+/C) and non-callipyge phenotype (+/+, C/+, and C/C) lambs were profiled to provide insight into the biochemical changes affecting meat quality attributes. M. longissimus thoracis from lambs with all four possible callipyge genotype (n = 4, C/+, C/C, +/C, and +/+) were collected after 3d aging and analyzed using mass-spectrometry based platforms. Among identified proteomes, cytochrome c (pro-apoptotic protein) was detected with significantly lower abundances in +/C. Anti-apoptotic HSP70, BAG3, and PARK7 were over-abundant in +/C, which could result in delayed apoptosis and possibly attributed to tougher meat in callipyge lambs. Eight glycolysis enzymes were overabundant in +/C lambs, whereas 3 enzymes involved in TCA cycle were overabundant in non-callipyge ones (C/C and/or C/+). Twenty-five metabolites were affected by genotypes (P < .05), including metabolic co-factors, polyphenols, and AA/short peptides. Our omics results provided insightful information for revealing the differences in biochemical attributes caused by callipyge mutation.

Polymorphism of CLPG gene in three sheep breeds grown in the steppe zone of the Russian Federation.

OBJECTIVE: This study aims to investigate the callipyge gene (CLPG) polymorphism in sheep of Edilbay, Volgograd, and Kalmyk breeds. MATERIALS AND METHODS: The analysis was performed by the polymerase chain reaction-restriction fragment length polymorphisms method. The objects of the study were Edilbay fat-tailed sheep (n = 500) at the breeding plant Volgograd-Edilbay (Volgograd region), Volgograd fine-wool sheep (n = 500) at the breeding plant Romashkovskiy (Volgograd region), and Kalmyk fat-tailed sheep (n = 500) at the breeding plant Kirovsky (the Republic of Kalmykia, Yashkul rayon). To conduct the research, tissue samples of 1 cm² from sheep of Kalmyk and Edilbay breeds were taken from the auricle. RESULTS: The allelic CLPG gene variants have been determined and genotypes of representative sampling of the three breeds of livestock grown in the steppe zone of Russia. The presented results of the CLPG gene polymorphism in these sheep breeds grown in Russia were obtained for the first time. The research study has revealed that in terms of the CLPG gene, the Edilbay, Volgograd, and Kalmyk sheep breeds have only a homozygous form. CONCLUSION: The results obtained expand the current understanding of the molecular markers that characterize the meat qualities of sheep.

Growth performance and meat characteristics of the first filial Awassi-Rambouillet callipyge ram lambs.

AIM: This study was designed to introduce the callipyge (CLPG) and 50% of Rambouillet sheep genes to improve meat quality and quantity of Awassi (AW) sheep. MATERIALS AND METHODS: The CLPG mutation was introduced into the AW sheep through frozen semen of homozygous Rambouillet rams for the CLPG mutation. Four ram lambs from the first-generation Rambouillet callipyge Awassi (F1-RCA) and five from pure AW were recruited for a fattening trial conducted in individual pens using standard ration, following which ram lambs were slaughtered for carcass and meat evaluation. RESULTS: Final body weight, dry matter intake, average daily gain, and feed conversion ratio were significantly higher in F1-RCA than AW. Hot and cold carcass weights and the other carcass cuts' weights, except for the fat tail, were heavier in F1-RCA than AW. There was no difference in dressing percentage between the two genetic groups (p>0.05). All non-carcass components' weights, except spleen, kidney, and testis, were higher in F1-RCA. Total lean, total bone, and intermuscular fat weight were greater in F1-RCA, but bone-to-lean ratio was lower in F1-RCA when compared with AW (p<0.01). No differences (p>0.05) were observed in all meat quality parameters for muscle longissimus with the exception of pH, redness color, and tenderness that were lower (p<0.05) in F1-RCA than AW. F1-RCA lambs had larger longissimus muscle area (30.9 vs. 16.9 cm2) and less leg fat depth (11.1 vs. 17.4 mm). CONCLUSION: The implications of this research show the potential of 50% of Rambouillet genes and the CLPG mutation to improve growth and meat characteristics in AW-Rambouillet crosses and can be used further to develop a meat-type AW with improved productivity and muscle mass.

Identification of genes directly responding to DLK1 signaling in Callipyge sheep.

BACKGROUND: In food animal agriculture, there is a need to identify the mechanisms that can improve the efficiency of muscle growth and protein accretion. Callipyge sheep provide excellent machinery since the up-regulation of DLK1 and RTL1 results in extreme postnatal muscle hypertrophy in distinct muscles. The aim of this study is to distinguish the genes that directly respond to DLK1 and RTL1 signaling from the genes that change as the result of muscle specific effects. RESULTS: The quantitative PCR results indicated that DLK1 expression was significantly increased in hypertrophied muscles but not in non-hypertrophied muscles. However, RTL1 was up-regulated in both hypertrophied and non-hypertrophied muscles. Five genes, including PARK7, DNTTIP1, SLC22A3, METTL21E and PDE4D, were consistently co-expressed with DLK1, and therefore were possible transcriptional target genes responding to DLK1 signaling. Treatment of myoblast and myotubes with DLK1 protein induced an average of 1.6-fold and 1.4-fold increase in Dnttip1 and Pde4d expression respectively. Myh4 expression was significantly elevated in DLK1-treated myotubes, whereas the expression of Mettl21e was significantly increased in the DLK1-treated myoblasts but reduced in DLK1-treated myotubes. DLK1 treatment had no impact on Park7 expression. In addition, Park7 and Dnttip1 increased Myh4 and decreased Myh7 promoter activity, resemble to the effects of Dlk1. In contrast, expression of Mettl21e increased Myh7 and decreased Myh4 luciferase activity. CONCLUSION: The study provided additional supports that RTL1 alone was insufficient to induce muscle hypertrophy and concluded that DLK1 was likely the primary effector of the hypertrophy phenotype. The results also suggested that DNTTIP1 and PDE4D were secondary effector genes responding to DLK1 signaling resulting in muscle fiber switch and muscular hypertrophy in callipyge lamb.

A new insight into meat toughness of callipyge lamb loins - The relevance of anti-apoptotic systems to decreased proteolysis.

The objective of this study was to determine associations of small heat shock proteins (sHSPs) in tenderness development of loins from callipyge and normal genotype lambs. Loins (M. longissimus lumborum) from sixteen lambs across four genotypes were collected throughout 9 days of postmortem aging. The loins from callipyge lambs had more intact desmin and troponin T throughout aging periods, as well as less µ-calpain autolysis and more calpastatin compared to loins from other genotypes (P < 0.05). Delayed onset of apoptosis was found in the callipyge loins indicated by less cytochrome c and more inactive procaspase-3 compared to normal lamb loins (P < 0.05). Less degraded HSP27 was also consistently found in the callipyge loins compared with loins from normal lambs (P < 0.001). The results found up-regulation of anti-apoptotic activities coincided with toughness in callipyge loins, which suggest apoptosis is likely involved in postmortem proteolysis and subsequent meat tenderization.

Myostatin-deficiency in mice increases global gene expression at the Dlk1-Dio3 locus in the skeletal muscle.

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth and development. Myostatin inhibition leads to increased skeletal muscle mass in mammals; hence, myostatin is considered a potential therapeutic target for skeletal muscle wasting. However, downstream molecules of myostatin in the skeletal muscle have not been fully elucidated. Here, we identified the Dlk1-Dio3 locus at the mouse chromosome 12qF1, also called as the callipyge locus in sheep, as a novel downstream target of myostatin. In skeletal muscle of myostatin knockout mice, the expression of mature miRNAs at the Dlk1-Dio3 locus was significantly increased. The increased miRNA levels are caused by the transcriptional activation of the Dlk1-Dio3 locus, because a significant increase in the primary miRNA transcript was observed in myostatin knockout mice. In addition, we found increased expression of coding and non-coding genes (Dlk1, Gtl2, Rtl1/Rtl1as, and Rian) at the Dlk1-Dio3 locus in myostatin-deficient skeletal muscle. Moreover, epigenetic changes, associated with the regulation of the Dlk1-Dio3 locus, were observed in myostatin knockout mice. Taken together, this is the first report demonstrating the role of myostatin in regulating the Dlk1-Dio3 (the callipyge) locus in the skeletal muscle.

Regulation of DLK1 by the maternally expressed miR-379/miR-544 cluster may underlie callipyge polar overdominance inheritance.

Inheritance of the callipyge phenotype in sheep is an example of polar overdominance inheritance, an unusual mode of inheritance. To investigate the underlying molecular mechanism, we profiled the expression of the genes located in the Delta-like 1 homolog (Dlk1)-type III iodothyronine deiodinase (Dio3) imprinting region in mice. We found that the transcripts of the microRNA (miR) 379/miR-544 cluster were highly expressed in neonatal muscle and paralleled the expression of the Dlk1. We then determined the in vivo role of the miR-379/miR-544 cluster by establishing a mouse line in which the cluster was ablated. The maternal heterozygotes of young mutant mice displayed a hypertrophic tibialis anterior muscle, extensor digitorum longus muscle, gastrocnemius muscle, and gluteus maximus muscle and elevated expression of the DLK1 protein. Reduced expression of DLK1 was mediated by miR-329, a member of this cluster. Our results suggest that maternal expression of the imprinted miR-379/miR-544 cluster regulates paternal expression of the Dlk1 gene in mice. We therefore propose a miR-based molecular working model for polar overdominance inheritance.

New insights into polar overdominance in callipyge sheep.

The callipyge phenotype in sheep involves substantial postnatal muscle hypertrophy and other changes to carcass composition. A single nucleotide polymorphism in the DLK1-DIO3 imprinted gene cluster alters gene expression of the paternal allele-specific protein-coding genes and several maternal allele-specific long noncoding RNA and microRNA when the mutation is inherited in cis. The inheritance pattern of the callipyge phenotype is polar overdominant because muscle hypertrophy only occurs in heterozygous animals that inherit a normal maternal allele and the callipyge SNP on the paternal allele (+/C). We examined the changes of gene expression of four major transcripts from the DLK1-DIO3 cluster and four myosin isoforms during the development of muscle hypertrophy in the semimembranosus as well as in the supraspinatus that does not undergo hypertrophy. The homozygous (C/C) animals had an intermediate gene expression pattern for the paternal allele-specific genes and two myosin isoforms, indicating a biological activity that was insufficient to change muscle mass. Transcriptome analysis was conducted by RNA sequencing in the four callipyge genotypes. The data show that homozygous animals (C/C) have lower levels of gene expression at many loci relative to the other three genotypes. A number of the downregulated genes are putative targets of the maternal allele-specific microRNA with gene ontology, indicating regulatory and cell signaling functions. These results suggest that the trans-effect of the maternal noncoding RNA and associated miRNA is to stabilize the expression of a number of regulatory genes at a functional, but low level to make the myofibers of homozygous (C/C) lambs less responsive to hypertrophic stimuli of the paternal allele-specific genes.

Genes contributing to genetic variation of muscling in sheep.

Selective breeding programs aiming to increase the productivity and profitability of the sheep meat industry use elite, progeny tested sires. The broad genetic traits of primary interest in the progeny of these sires include skeletal muscle yield, fat content, eating quality, and reproductive efficiency. Natural mutations in sheep that enhance muscling have been identified, while a number of genome scans have identified and confirmed quantitative trait loci (QTL) for skeletal muscle traits. The detailed phenotypic characteristics of sheep carrying these mutations or QTL affecting skeletal muscle show a number of common biological themes, particularly changes in developmental growth trajectories, alterations of whole animal morphology, and a shift toward fast twitch glycolytic fibers. The genetic, developmental, and biochemical mechanisms underpinning the actions of some of these genetic variants are described. This review critically assesses this research area, identifies gaps in knowledge, and highlights mechanistic linkages between genetic polymorphisms and skeletal muscle phenotypic changes. This knowledge may aid the discovery of new causal genetic variants and in some cases lead to the development of biochemical and immunological strategies aimed at enhancing skeletal muscle.