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The intramuscular fat content plays a crucial role in meat quality traits. Increasing the degree of adipogenesis in beef cattle leads to an increase in the content of intramuscular fat. Adipogenesis a complex biochemical process which is under firm genetic control. Over the last three decades, the Qinchuan beef cattle have been extensively studied for the improvement of meat production and quality traits. In this study, we reviewed the literature regarding adipogenesis and intramuscular fat deposition. Then, we summarized the research conducted on the transcriptional regulation of key adipogenic marker genes, and also reviewed the roles of adipogenic marker genes in adipogenesis of Qinchuan beef cattle. This review will elaborate our understanding regarding transcriptional regulation which is a vital physiological process regulated by a cascade of transcription factors (TFs), key target marker genes, and regulatory proteins. This synergistic action of TFs and target genes ensures the accurate and diverse transmission of the genetic information for the accomplishment of central physiological processes. This information will provide an insight into the transcriptional regulation of the adipogenic marker genes and its role in bovine adipogenesis for the breed improvement programs especially for the trait of intramuscular fat deposition.

Perilipin 1 (PLIN1) protein, also known as lipid droplet-associated protein, is encoded by the PLIN1 gene and is able to anchor itself to the membranes of lipid droplets. The phosphorylation of PLIN1 is critical for the mobilization of fat in adipose tissue and plays an important role in regulating lipolysis and lipid storage in adipocytes. However, research on the synthesis and lipid metabolism of lipid droplets by PLIN1 in bovine adipocytes is limited. In the present study, we found that bovine PLIN1 was highly expressed in subcutaneous adipose tissue. The highest level of PLIN1 mRNA expression in bovine adipocytes was observed on day 6 of differentiation. Moreover, the cytoplasmic subcellular localization of PLIN1 was observed in bovine preadipocytes. To elucidate the molecular mechanism of bovine PLIN1 transcriptional regulation, we cloned eight fragments containing the 5′ regulatory region of the PLIN1 gene. The results showed that the −209/−17 bp region of the bovine PLIN1 gene was the core promoter region. Based on the transcriptional activities of the promoter vector fragments, the luciferase activity of the mutated fragment, the siRNA interference, and the results of the electrophoretic mobility shift assay (EMSA), we identified the binding sites of E2F transcription factor 1 ( E2F1), pleiomorphic adenoma gene 1 (PLAG1 ), CCAAT enhancer binding protein beta ( C/EBPβ ), and SMAD family member 3 ( SMAD3 ) as the transcriptional activators or repressors of the core promoter region. Further experiments confirmed that the knockdown of the PLIN1 gene affected the ability of these transcription factors to regulate the lipid metabolism in bovine adipocytes. In conclusion, our results reveal a potential mechanism for the transcriptional regulation of PLIN1 in bovine adipocytes.

The TORC2 gene is a member of the transducer of the regulated cyclic adenosine monophosphate (cAMP) response element binding protein gene family, which plays a key role in metabolism and adipogenesis. In the present study, we confirmed the role of TORC2 in bovine preadipocyte proliferation through cell cycle staining flow cytometry, cell counting assay, 5-ethynyl-2&prime, deoxyuridine staining (EdU), and mRNA and protein expression analysis of proliferation-related marker genes. In addition, Oil red O staining analysis, immunofluorescence of adiponectin, mRNA and protein level expression of lipid related marker genes confirmed the role of TORC2 in the regulation of bovine adipocyte differentiation. Furthermore, the transcription start site and sub-cellular localization of the TORC2 gene was identified in bovine adipocytes. To investigate the underlying regulatory mechanism of the bovine TORC2, we cloned a 1990 bp of the 5' untranslated region (5&prime, UTR) promoter region into a luciferase reporter vector and seven vector fragments were constructed through serial deletion of the 5&prime, UTR flanking region. The core promoter region of the TORC2 gene was identified at location &minus, 314 to &minus, 69 bp upstream of the transcription start site. Based on the results of the transcriptional activities of the promoter vector fragments, luciferase activities of mutated fragments and siRNAs interference, four transcription factors (CCAAT/enhancer-binding protein C/BEP, X-box binding protein 1 XBP1, Insulinoma-associated 1 INSM1, and Zinc finger protein 263 ZNF263) were identified as the transcriptional regulators of TORC2 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. Furthermore, we also identified that C/EBP, XBP1, INSM1 and ZNF263 regulate TORC2 gene as activators in the promoter region. We can conclude that TORC2 gene is potentially a positive regulator of adipogenesis. These findings will not only provide an insight for the improvement of intramuscular fat in cattle, but will enhance our understanding regarding therapeutic intervention of metabolic syndrome and obesity in public health as well.

Intramuscular fat is the an important factor that defines meat quality; however, enhancing its deposition without increasing the other three adipose depots (subcutaneous, visceral, and intermuscular fat) is a challenge for animal science and the meat industry. The TORC1 is a key regulator of adipogenesis and its regulation in bovine intramuscular preadipocytes has not been studied. The TORC1 is a member of the gene family that codes for a binding proteins which regulate transcription of cAMP which, is a key regulator of adipogenesis. In the present study, expression and sub-cellular localization of the TORC1 gene was analyzed in bovine preadipocytes. Bioinformatics tools were applied to characterize TORC1. To investigate the molecular mechanism of bovine TORC1 gene regulation, we cloned a 1008 bp of the 5’UTR regulatory region into a luciferase reporter vector. Different fragments were amplified using 5’UTR unidirectional deletion of the TORC1 promoter. Site directed mutation, dual luciferase reporter assay, RNAi interference and DNA-protein interaction (EMSA) were used to validate the regulatory roles of Smad3 and NRF1 in the regulation of TORC1 gene in bovine preadipocytes. The core promoter region of the TORC1 gene was identified at a location −410 to −155 bp upstream of transcription start site. Different vectors were constructed through serial deletion of the 5’UTR flanking region and in combination with site directed mutations and transcription interference through siRNA or shRNA, two transcription factors of NRF1 and Smad3 were found to be repressors in the promoter of the TORC1 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. The core promoter region of the TORC1 gene, spanning from −410 to −155 bp upstream of the transcription start site was specified in this study and this information will provide opportunity for the improvement of intramuscular fat in cattle.

Pre-slaughter and slaughter stressors are considered major concerns in animal welfare. Halal slaughtering method is considered one of the slaughtering stressors in livestock. This method seems to cause fear followed by stress in animals mainly due to inhuman handling. In this review, empathy and animal welfare are discussed in light of Islamic sharia and has further linked with animal's physiology and behavioral responses during slaughtering. Islam as a religion forbids slaughtering an animal in front of another animal as through optic, olfactory, and cochlear senses animals can perceive the stress state of conspecifics. This suggests and strengthens the hypothesis that animals being slaughtered in front of each other may produce stress in them. This argument further leads to a claim that animals can experience empathy of each other through olfaction of semiochemicals (stress pheromones) emitted from animals slaughtered in the stressful condition that can be detected by other animals in abattoirs. Hence, research is needed to find out these specific stress pheromones and legislation needs to be adopted in slaughterhouses to isolate the areas of butchery from slaughtering lines to ensure proper guidelines of Halal slaughtering in slaughterhouses.

CCAAT/enhancer-binding protein beta (C/EBPβ), as an essential transcriptional factor, regulates the differentiation of adipocytes and the deposition of fat. Herein, we cloned the whole open reading frame (ORF) of bovine C/EBPβ gene and analyzed itsputative protein structures via DNA cloning and sequence analysis. Then, theexpression profile of this C/EBPβ gene in fifteen kinds of tissues of Qinchuan cattlewas conducted by real-time polymerase chain reaction (RT-PCR) technology. One significant outside to inside transmembrane structure located in amino acid region from 190 to 208 was observed in the putative protein sequences. Besides, one basic leucine zipper domain (bZIP) in amino acid area from 274 to 337 was found, concurring with the main characteristic of C/EBPs. Homologous comparison of the amino acid sequences from C/EBPβ cloned in this study and those from different species indicated C/EBPβ gene of Qinchuan cattle shared 97, 95 and 91% similarity with Homo sapiens, Sus scrofa and Oryctolagus cuniculus respectively, indicating a good sequence evolutional conservation of C/EBPβ. RT-PCR results revealed bovine C/EBPβ gene mRNA expression level of subcutaneous fat was the highest among all the analyzed tissues, and the relative quantity (RQ) in fat tissue increased as cattle grew. All in all, the present results could be used as basic but important genetic resource and information to inspire additional and specific studies on Qinchuan cattle. Key words: CCAAT/enhancer-binding protein beta, molecular cloning, expression analysis.