In the biological process of testicular spermatogenesis, the expression and interaction of many genes are regulated by microRNAs (miRNAs). However, comparisons of miRNA expression between descended testes (DTs) and undescended testes (UDTs) are rarely done in horses. In this study, we selected two UDTs (CKY2b and GU4b) from Chakouyi (CKY) and Guanzhong (GU) horses and eight DTs (GU1–3, CKY1, CKY3, CKY2a, GU4a, and GU5). Three groups were compared to evaluate expression patterns of testicular miRNA in stallion testes. Group 1 compared normal CKY horses and GU horses (CKY1 and CKY3 vs. GU1–3). Group 2 (CKY2a and GU4a (DTs) vs. CKY2b and GU4b (UDTs)) and group 3 (GU1–3, CKY1, CKY3 (DTs) vs. CKY2b and GU4b (UDTs)) compared the expression levels in unilateral retained testes to normal testes. The results show that 42 miRNAs (7 upregulated and 35 downregulated) had significantly different expression levels in both comparisons. The expression levels of eca-miR-545, eca-miR-9084, eca-miR-449a, eca-miR-9024, eca-miR-9121, eca-miR-8908e, eca-miR-136, eca-miR-329b, eca-miR-370, and eca-miR-181b were further confirmed by quantitative real-time PCR assay. The target genes of differentially expressed miRNAs in three comparisons were predicted, and the functions were annotated. The putative target genes of the 42 co-differentially expressed miRNAs were annotated to 15 functional terms, including metal ion binding, GTPase activator activity, zinc ion binding, intracellular, cytoplasm, and cancer pathways, and osteoclast differentiation. Our data indicate that the differentially expressed miRNAs in undescended testis suggests a potential role in male fertility and a relationship with cryptorchidism in horses. The discovery of miRNAs in stallion testes might contribute to a new direction in the search for biomarkers of stallion fertility.

Testes produce sperm, and investigations into gene expression in the testes will enhance the understanding of the roles of testicular genes in male reproduction. Cryptorchidism, the failure of one or both testes to descend into the scrotal sac, is a common congenital malformation in horses. The major clinical consequence of this abnormality is impaired fertility. The aim of this study was to analyze the expression patterns of testicular genes and to identify the differentially expressed genes (DEGs) in testes between cryptorchid and normal horses. In this study, the gene expression patterns in equine testes and the DEGs between mature descended testes (DTs) and undescended testes (UDTs) were identified by RNA-seq and validated by real-time qPCR. Our results provide comprehensive transcriptomic data on equine testes. The transcriptomic analysis revealed 11 affected genes that were downregulated in UDTs, possibly as a result of the higher temperature in the abdomen than in the scrotal sac. These 11 genes have previously been associated with male reproduction, and their downregulation might explain the impaired fertility of cryptorchid horses. Two homozygous missense mutations detected in horses with cryptorchidism were absent in normal horses and were listed as potential pathogenic mutations; these mutations should be verified in the future.

Chinese herbal medicine additives (CHMAs) have become increasingly popular as sustainable alternatives to synthetic compounds for improving the quality of mutton. However, the precise molecular mechanisms underlying their effects are not well understood. By integrating transcriptomic profiling, metabolomic pathways, and microbial community dynamics, this review deciphers the synergistic mechanisms of CHMAs in enhancing mutton flavor, supported by empirical evidence from 2014 to 2024. Our key findings highlight three synergistic pathways: (1) Epigenetic suppression of FASN and CYP2B6 via DNA methylation leads to a reduction of odor precursors like 4-methyloctanoic acid by 30–50% (p < 0.01); (2) Mulberry leaf flavonoids activate β-oxidation, increasing linoleic acid content by 25%, thereby improving tenderness and juiciness; (3) Licorice polysaccharides, in collaboration with Ruminococcus-enriched microbiota, enhance flavor volatiles such as 2-acetylthiazoline. It is important to consider dose-dependent thresholds, as thyme phenolic extract at 0.05% maximizes aroma intensity (p < 0.05), while exceeding 1.5% licorice glycyrrhizin intensifies gaminess. Species-specific responses highlight variations in rumen microbial activity, with Tan sheep showing a 30% increase in catalase activity compared to goats. Validated by the Luoyang Longxupo industrial model, which achieved a 30% reduction in odor and received Green Food Certification, this study proposes a unique gene-metabolite-microbe interaction network that emphasizes the significance of epigenetic-microbial crosstalk. We also discuss challenges related to herbal synergies, sensory standardization, and offer solutions through AI-driven optimization, with an AUC of 0.89, as well as the potential application of cultured meat, such as Salvia miltiorrhiza reducing lipid oxidation by 40% in vitro. These findings connect traditional herbal knowledge with precision agriculture, providing practical strategies for environmentally friendly mutton production that meets the global demand for safe, high-quality protein.

MicroRNAs (miRNAs) are small RNA molecules consisting of approximately 22 nucleotides that are engaged in the regulation of various bio-processes. There is growing evidence that miR-29 is a key regulator of hepatic lipid metabolism. Mimics and inhibitors of bta-miRNA29d-3p were transiently transfected in bovine mammary epithelial cells (BMECs) to reveal the regulation of bta-miRNA29d-3p on lipid accumulation in BMECs. Results showed that overexpression of bta-miRNA29d-3p significantly inhibited the expression of genes related to triglyceride (TAG) synthesis, namely DGAT1 and mitochondrial glycerol-3-phosphate acyltransferase (GPAM, p < 0.01) and down-regulated TAG levels in cells (p < 0.05). The expression of fatty acid synthesis and desaturation-related genes FASN, SCD1, and ACACA, and transcription factor SREBF1 also decreased. Interference of bta-miRNA29d-3p significantly increased the expression of GPAM, DGAT1, FASN, SCD1, ACACA, and SREBF1 (p < 0.01), and significantly upregulated the concentration of TAG in cells. Furthermore, a luciferase reporter assay confirmed that GPAM is a direct target of bta-miRNA29d-3p. In summary, bta-miRNA29d-3p modulates fatty acid metabolism and TAG synthesis by regulating genes related to lipid metabolism in BMECs and targeting GPAM. Thus, bta-miRNA29d-3p plays an important role in controlling mammary lipid synthesis in cows.

To address the bottleneck in meat production efficiency within China’s mutton sheep industry, this study established a two-generation crossbreeding program between WHS rams and STH ewes. Hybrid offspring (F1 and BC1) were evaluated for growth performance, slaughter traits, meat quality, and reproductive performance. The F1 generation exhibited significant improvements over STH in 6-month body weight (52.3 kg, +27.3%), dressing percentage (56.1%, +6.3%), and feed efficiency ratio (FER) of 4.6 (p < 0.05). The BC1 generation shows a mixed state of paternal and maternal characteristics and further enhanced these traits, achieving a 6-month body weight of 55.2 kg (+5.4% vs. F1), a dressing percentage of 58.3%, and an optimized gain-to-feed ratio (G:F) of 4.2 (−8.7%). Meat quality parameters, including shear force (32.5 N vs. 41.6 N in F1 vs. STH) and intramuscular fat content (4.5% vs. 3.8% in F1 vs. STH), demonstrated superior tenderness and marbling. Despite a decline in lambing rate (F1: 178%; BC1: 142%), the hybrids combined the dam’s adaptability with the sire’s meat traits, forming a novel germplasm for sustainable mutton production. This study provides a replicable model for balancing genetic improvement and ecological sustainability in central China.