Transcriptome-Metabolome Analysis Reveals That Crossbreeding Improves Meat Quality in Hu Sheep and Their F1-Generation Sheep

Abstract

Consumers are increasingly demanding higher-quality mutton. Crossbreeding has been recognized as an effective means to improve meat quality. However, the phenomenon underlying these molecular system mechanisms remains largely unidentified. In this study, 48 male lambs aged 3 months were selected, including ♂ Hu sheep × ♀ Hu (HH, n = 16), ♂ Polled Dorset × ♀ Hu sheep F₁ hybrid lambs (DH, n = 16), and ♂ Southdown × ♀ Hu sheep (SH, n = 16) F₁ hybrid lambs, and raised in a single pen under the same nutritional and management conditions for 95 days. Then, seven sheep close to the average weight of the group were selected and fasted for 12 h prior to slaughter. By comparing the muscle fiber characteristics of the Longissimus dorsi of the three groups of sheep, and through transcriptomic and metabolomic analyses, we revealed molecular differences in the meat quality of Hu sheep crossbred with different parent breeds. The results of this study showed that muscle fiber diameter and cross-sectional area were significantly greater in the DH group than in the HH group, and collagen fiber content in the DH group was also significantly higher than in the HH group (p < 0.05). A total of 163 differential genes and 823 differential metabolites were identified in the three groups, most of which were related to muscle development and lipid metabolism. These included the AMPK signaling pathway, the PI3K-Akt signaling pathway, glycerophospholipid metabolism, and the related genes EFHB, PER3, and PPARGC1A. The results of this study offer valuable insights into the molecular mechanisms underlying the impact of crossbreeding on meat quality and provide a theoretical foundation for sheep crossbreed production.

Keywords: crossbreed; longissimus dorsi; meat quality; metabolomics; transcriptomics.

Figure 1

Muscle fiber morphology traits in the HH, DH, and SH groups. (A) Tissue sections of the longissimus dorsi muscle were stained with HE (5× magnification). (B–D) Comparison of muscle fiber density, muscle fiber cross-sectional area, and muscle fiber diameter among the three groups. All data are expressed as mean ± SD (n = 7). Different letters indicate significant differences (p < 0.05). HH: ♂ Hu × ♀ Hu sheep; DH: ♂ Polled Dorset × ♀ Hu sheep; SH: ♂ Southdown × ♀ Hu sheep.

Figure 2

Collagen fiber content in the HH, DH, and SH groups using Masson staining. (A–C) Tissue sections of dorsal longissimus muscle were stained with Masson (5× magnification). (D) Comparison of collagen fiber content among the three groups. All data are expressed as mean ± SD (n = 7). Different letters indicate significant differences (p < 0.05). HH: ♂ Hu × ♀ Hu sheep; DH: ♂ Polled Dorset × ♀ Hu sheep; SH: ♂ Southdown × ♀ Hu sheep.

Figure 3

Muscle fiber type composition in the HH, DH, and SH groups. (A) Immunofluorescence staining detected the distributions of fast and slow muscle fibers. (B) The numerical proportion of fast and slow fibers in the muscle. All data are expressed as mean ± SD (n = 7). Different letters indicate significant differences (p < 0.05). HH: ♂ Hu × ♀ Hu sheep; DH: ♂ Polled Dorset × ♀ Hu sheep; SH: ♂ Southdown × ♀ Hu sheep.

Figure 4

Transcriptomic comparisons of the longissimus dorsi for the DH vs. HH, SH vs. DH, and SH vs. HH groups. (A) Venn plot of three groups. (B) Heatmap of differential gene clustering. (C) Volcano plots of the DEGs. Red dots represent up-regulated DEGs; green dots represent down-regulated DEGs.

Figure 5

Functional enrichment analysis of the longissimus dorsi samples of the DH vs. HH, SH vs. DH, and SH vs. HH groups. (A) DEG GO enrichment histogram. The horizontal axis indicates the number of differential genes annotated to the entry, and the vertical axis indicates the name of the GO entry. The numbers in the figure indicate the number of differential genes annotated to the entry, the ratio of the number of differential genes annotated to the GO entry to the total number of differential genes is shown in parentheses, and the label on the far right represents the classification to which the GO entry belongs. (B) KEGG enrichment scatter plot of DEGs. The horizontal axis represents the Rich factor; the larger the Rich factor, the greater the degree of enrichment. The vertical axis represents the KEGG pathway; the larger the point, the greater the number of differential genes enriched in the pathway; the redder the color of the point, the more significant the enrichment. (C) Gene set enrichment analysis of the DH vs. HH, SH vs. DH, and SH vs. HH groups. The path name in the figure has been abbreviated, please refer to the complete name of the Supplementary Materials.

Figure 6

LC-MS/MS analysis of longissimus dorsi metabolic profiles for the DH vs. HH, SH vs. DH, and SH vs. HH groups. (A) OPLS-DA Score Chart. (B–D) DM volcano map.

Figure 7

DM KEGG classification map of the DH vs. HH, SH vs. DH, and SH vs. HH groups. The vertical axis is the name of the KEGG metabolic pathway, and the horizontal axis is the number of DMs annotated to the pathway and the ratio of that number to the total number of differential metabolites for which they were annotated. (A–C) KEGG enrichment bar chart of the DH vs. HH, SH vs. DH, and SH vs. HH groups.

Figure 8

Comprehensive analysis of transcriptomics and metabolomics. (A–C) KEGG enrichment analysis bar graph: horizontal axes represent the number of differential metabolites and DEGs enriched in the pathway, vertical axes represent KEGG pathway names, and red and green bars represent the metabolome and transcriptome, respectively. (D) Correlation network diagram: a green circle is the name of the DEG, a red circle is the name of the DM, the thickness of a line represents the level of correlation, a green line represents negative correlation, and a red line represents positive correlation (p < 0.05).

Figure 9

Validation of RNA-Seq by qRT-PCR analysis.