Improving meat quality and developing superior cattle breeds are ongoing priorities in livestock production. Intramuscular fat (IMF) content significantly influences beef quality, impacting flavor, tenderness, and overall palatability. Genetic factors play a crucial role in determining IMF deposition, making the identification of key genes and pathways critical for selective breeding programs. Shandong black cattle, a newly developed breed in China, is known for its superior meat quality, while Luxi cattle represent a well-established Chinese beef breed. This study aims to elucidate the genetic basis of the differences in fat deposition between these two breeds by employing high-throughput RNA sequencing (RNA-Seq) to analyze the transcriptome of their longissimus dorsi muscles. Understanding the molecular mechanisms regulating IMF content will inform the development of marker-assisted selection strategies and facilitate the genetic improvement of beef cattle. Previous research has utilized various methods, such as microarrays and quantitative PCR, to identify candidate genes involved in lipid metabolism in cattle. However, RNA-Seq offers a more comprehensive approach, allowing for simultaneous detection of the expression levels of thousands of genes, thereby providing a more holistic view of the gene regulatory networks involved in IMF deposition. By comparing the transcriptomes of Shandong black cattle and Luxi cattle, we aim to identify differentially expressed genes and pathways associated with differences in IMF, thereby providing valuable insights for targeted breeding programs and future research directions.

Several studies have investigated the genetic basis of fat deposition in cattle using various approaches. Zhang Yingying et al. (2010) used gene chip technology to identify 12 genes, including COL1A2, COL1A1, SPP1, MMP2, MYH3, and MYH8, as potential candidates affecting beef quality in Qinchuan cattle. McCabe et al. (2012) employed RNA-Seq to analyze differential gene expression in the liver of lactating dairy cows, linking negative regulation of liver production to fat metabolism. Bai et al. (2016) utilized RNA-Seq to compare whole blood transcriptional profiles in Chinese Holstein cows with different milk yields, highlighting the emerging role of high-throughput sequencing in livestock genomics. These studies demonstrate the power of transcriptomic analysis in unraveling the complexities of fat metabolism in cattle. However, there is a need for further investigation into the specific genes and pathways involved in intramuscular fat deposition, particularly in different breeds with varying IMF characteristics. This study will build upon these previous findings by focusing on the comparison between two specific breeds, Shandong black cattle and Luxi cattle, to provide more detailed insights into breed-specific differences in fat deposition.

This study utilized a comparative transcriptomics approach to investigate the genetic basis of fat deposition differences between Shandong black cattle and Luxi cattle. Sixteen-month-old healthy animals (n=3 per breed) were selected, ensuring uniformity in age and health status to minimize confounding factors. The longissimus dorsi muscle, a well-established indicator of beef quality, was collected immediately post-slaughter under strict sterile conditions to maintain RNA integrity. Fatty acid composition of the muscle samples was determined using gas chromatography (GC) after preparation of fatty acid methyl esters. RNA extraction was performed using Trizol reagent, ensuring removal of genomic DNA contamination using DNase I treatment. RNA quality was rigorously assessed using an Agilent 2100 Bioanalyzer and NanoDrop 2000 spectrophotometer, ensuring high-quality RNA (RIN ≥ 6.5, OD260/280 = 1.8-2.2, OD260/230 ≥ 2.0) for library preparation. Illumina HiSeq X Ten sequencing platform was employed for high-throughput sequencing, generating a large volume of sequence data. Raw sequencing data underwent quality control and filtering using Fastp software, removing low-quality reads, adapter sequences, and poly-N reads. Clean reads were aligned to the Bos taurus genome (ARS-UCD1.2) using HISAT2 and Bowtie2, ensuring accurate mapping and minimizing bias. Differentially expressed genes (DEGs) were identified using Cufflinks and Cuffdiff, setting stringent criteria (FPKM ≥ 1, |log2(fold change)| ≥ 2, q-value < 0.05) to minimize false positives. Gene Ontology (GO) enrichment analysis was performed using GOatools and clusterProfiler to identify functional categories enriched among DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using KOBAS to identify significantly enriched metabolic pathways. Protein-protein interaction (PPI) networks were constructed using STRING database and visualized using Cytoscape to examine relationships among DEGs. Quantitative real-time PCR (qRT-PCR) and Western blotting were employed to validate selected DEGs identified through RNA-Seq. Statistical analysis was performed using SPSS 17.0 for fatty acid analysis and appropriate R packages for transcriptomic data analysis. The study adhered to strict ethical guidelines, obtaining approval from the Committee on the Ethics of Animal Experiments of Qingdao Agricultural University.

The analysis revealed significant differences in fatty acid composition between the two breeds. Shandong black cattle exhibited a higher ratio of unsaturated fatty acids to saturated fatty acids (1.37:1) compared to Luxi cattle (1.24:1), indicating a more favorable fatty acid profile for meat quality. RNA-Seq identified 1320 DEGs between the two breeds. Gene Ontology (GO) analysis revealed enrichment of DEGs in several biological processes related to lipid metabolism, including regulation of the Wnt signaling pathway, fat cell differentiation, and cAMP metabolic processes. KEGG pathway analysis indicated significant enrichment in pathways such as regulation of lipolysis in adipocytes, the PPAR signaling pathway, and the AMPK signaling pathway. Network analysis pinpointed FABP4 and ADIPOQ as key genes connected to multiple metabolic pathways. These two genes shared seven common regulatory factors (PLIN1, PLIN2, PPARGC1A, RXRA, PCK1, LEPR, LEP) involved in lipolysis regulation, adipocytokine signaling, and PPAR signaling. qRT-PCR and Western blotting validated the RNA-Seq data, confirming the differential expression of these key genes. The correlation analysis showed a positive correlation between the expression levels of FABP4 and ADIPOQ and several growth traits, including body weight and chest circumference.

The findings of this study provide valuable insights into the molecular mechanisms underlying the differences in fat deposition between Shandong black cattle and Luxi cattle. The higher ratio of unsaturated to saturated fatty acids in Shandong black cattle aligns with its reputation for superior meat quality. The significant enrichment of DEGs in pathways related to lipid metabolism, particularly the PPAR and AMPK signaling pathways, highlights the central role of these pathways in regulating IMF deposition. The identification of FABP4 and ADIPOQ as key candidate marker genes offers a potential target for marker-assisted selection in beef cattle breeding programs. The shared regulatory factors between these two genes suggest a complex interplay of regulatory mechanisms governing fat metabolism. The validation of the RNA-Seq data using qRT-PCR and Western blotting strengthens the reliability of the findings. Future research could focus on investigating the functional roles of these genes and their regulatory networks in more detail, potentially through gene editing or other functional genomics techniques. The results of this study contribute to a deeper understanding of the genetic basis of fat deposition in beef cattle, paving the way for targeted breeding strategies to enhance meat quality.

This study employed RNA-Seq to analyze the transcriptome of longissimus dorsi muscle from Shandong black cattle and Luxi cattle, revealing significant differences in gene expression related to lipid metabolism. FABP4 and ADIPOQ emerged as key candidate genes for fat deposition, showing consistent differential expression across RNA-Seq, qRT-PCR, and Western blotting. These findings provide valuable molecular markers for marker-assisted selection and offer novel insights into the genetic control of intramuscular fat in beef cattle. Future research should focus on functional validation of these genes and exploring their potential as predictive markers for beef quality traits.

The sample size in this study was relatively small (n=3 per breed), which may limit the statistical power and generalizability of the findings. Further studies with larger sample sizes are needed to confirm the results and assess their broader applicability across different populations of cattle. The study focused on a single muscle (longissimus dorsi), and the findings may not be entirely representative of other muscle types or fat depots in the animal. Further investigation is needed to determine the extent to which these findings translate to other tissues and breeds.