Lipin-1 is known to play a regulatory role in tissues that function in lipid metabolism. In dairy cows, the lipin-1 gene (LPIN1) is highly expressed in the mammary gland, but its function in milk production is less understood. In this study, we used PCR-single strand conformation polymorphism analysis to investigate sequence variation in three regions of bovine LPIN1 in New Zealand Holstein-Friesian × Jersey (HF × J)-cross dairy cows, including part of the 5′ non-coding region, the region containing the LPIN1β-spliced exon, and the sixth coding exon that encodes the putative transcriptional activating domain of the protein. No variation was found in the LPIN1β-spliced exon, but two sequence variants containing one single nucleotide polymorphism (SNP) were identified in the 5′ non-coding region and four sequence variants containing four non-synonymous SNPs were identified in the sixth coding exon. Among the three common variants of the sixth coding exon, variant C was found to be associated with an increase in milk fat percentage (presence 4.96 ± 0.034% vs. absence 4.81 ± 0.050%; p = 0.006) and milk protein percentage (presence 4.09 ± 0.017% vs. absence 3.99 ± 0.025%; p = 0.001), but no associations (p > 0.01) were detected for milk yield. These results suggest that variation in LPIN1 affect the synthesis of fat and proteins in milk and has potential as a gene-marker to improve milk production traits.

Bovine and ovine respiratory diseases are commonly associated with multiple bacterial pathogens, including Mycoplasma bovis (Mb), Mycoplasma ovipneumoniae (Mo), Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), and Mycobacterium bovis (Tb). Among these, Mb and Mo are the primary causative agents of mycoplasma pneumonia in cattle and sheep, respectively. Based on this difference, two multiplex PCR assays were developed for the detection of bacterial respiratory pathogens in cattle and sheep, differing mainly in the primers targeting Mycoplasma spp. The bovine assay simultaneously detected Mb, Mh, Pm, and Tb, while the ovine assay detected Mo, Mh, Pm, and Tb, with expected amplicon sizes of 319/320 bp, 390 bp, 528 bp, and 274 bp. After optimization, both assays were able to simultaneously detect the target pathogens and showed good specificity, sensitivity, and reproducibility, with detection limits of 1.0 × 10³ copies/μL for the bovine assay and 1.0 × 10²–10³ copies/μL for the ovine assay. Preliminary testing using clinical samples demonstrated that the assays could detect the target bacterial pathogens. These results indicate that the developed multiplex PCR assays provide a basis for further validation and potential application in the detection of bacterial respiratory pathogens in cattle and sheep.

The intestinal metabolites of Tibetan sheep play a vital role in the integrated regulation of the host and the microbe-gut-brain axis. The current study sought to investigate the characteristics of alterations in rectal metabolites and their functional implications during the cold and warm seasons in Tibetan sheep. A cohort of 12 ewes, approximately 1 week ± 1 month in age, exhibiting good body condition and a similar genetic background, was selected for metabolomic analysis of rectal contents collected during the warm season (August) and the cold season (December). The findings revealed significant differences in the rectal microbial metabolites of Tibetan sheep between the two seasons (P < 0.05), with a total of 476 differential metabolites identified in the positive ion mode (148 up-regulated and 328 down-regulated) and 383 differential metabolites in the negative ion mode (135 up-regulated and 248 down-regulated). These differential metabolites were mapped to 12 KEGG metabolic pathways (P &lt; 0.05), including fatty acid biosynthesis, arachidonic acid metabolism, secondary bile acid biosynthesis, propionic acid metabolism, lysine degradation, and arginine and proline metabolism, which are linked to lipid metabolism, carbohydrate metabolism, and amino acid metabolism, respectively. The content of deoxycholic acid in the intestinal tract of Tibetan sheep was significantly higher during the cold season compared to the warm season (P &lt; 0.05), while propionic acid was significantly lower (P &lt; 0.05). These metabolites are involved in secondary bile acid biosynthesis and propanoate metabolism pathways. These results indicate significant seasonal variations in rectal microbial metabolites in Tibetan sheep. The identified metabolites may play a crucial role in regulating energy metabolism, inflammatory responses, and immune functions, thereby enhancing the adaptability of Tibetan sheep to the challenges posed by cold-season conditions.

Background: Most studies of high-altitude adaptation in animals have focused on the heart, lung, blood, and skeletal muscle, whereas systematic evidence regarding metabolism-related expression changes in the pituitary remains limited. The pituitary is an energy-demanding neuroendocrine organ characterized by active glucose utilization and lactate production. However, multi-omics changes associated with glycolysis–lactate metabolism in the pituitary of high-altitude sheep remain undefined. Methods: Pituitary tissues from adult female sheep living at high altitude were compared with those from adult female sheep living at low altitude (n = 3 per group). Transcriptomic differences were profiled by RNA sequencing, and proteomic differences were characterized by data-independent acquisition (DIA) mass spectrometry (MS). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, gene set enrichment analysis, and integrated transcriptomic–proteomic analyses were used to identify representative candidates, which were further validated by reverse transcription quantitative polymerase chain reaction and western blotting. Results: RNA sequencing identified 1222 differentially expressed genes, including 690 upregulated and 532 downregulated genes, whereas DIA-MS identified 2046 differentially expressed proteins, including 1096 upregulated and 950 downregulated proteins. Functional analyses indicated enrichment of glycolysis, lactate metabolism, pyruvate metabolism, the HIF-1 signaling pathway, and oxidative phosphorylation. Gene set enrichment analysis showed significant positive enrichment of oxidative phosphorylation in the high-altitude group, whereas glycolysis/gluconeogenesis showed a positive trend without meeting the false discovery rate threshold. Integrated transcriptomic–proteomic analysis identified hexokinase 2 (HK2), enolase 2 (ENO2), and lactate dehydrogenase A (LDHA) as concordantly upregulated at both the transcript and protein levels (q-value < 0.05). Reverse transcription quantitative PCR (RT-qPCR) showed significant upregulation of HK2, ENO2, and LDHA (all p < 0.001), while western blotting confirmed increased protein expression, with HK2 reaching p < 0.001 and ENO2 and LDHA reaching p < 0.01. Conclusions: HK2, ENO2, and LDHA are stably upregulated in the pituitary of high-altitude sheep, indicating remodeling of key expression nodes associated with glycolysis–lactate metabolism. These findings provide multi-omics evidence for high-altitude-associated remodeling of selected metabolic expression nodes in the sheep pituitary.

Tibetan sheep, a vital livestock species adapted to the extreme hypoxia, low temperatures, and intense radiation of the Qinghai–Tibet Plateau, rely on gastrointestinal microbiota for ecological balance and host nutrition, metabolism, and immunity. However, the possible associations of gut microbiota and metabolites with seasonal phenology remain unclear. Integrating biochemical, metagenomic, and metabolomic analyses, this study investigated seasonal variations in serum indices, microbial communities, and metabolites to inform enhanced breeding strategies. Analysis of forage nutritional composition showed that warm-season forages had significantly higher concentrations of dry matter (DM), crude protein (CP), and ether extract (EE) ( p < 0.01), whereas cold-season forages were characterized by significantly greater levels of neutral detergent fiber (NDF) and acid detergent fiber (ADF) ( p < 0.01). Correspondingly, serum analysis revealed significantly higher warm-season concentrations of alanine aminotransferase, total cholesterol, creatinine, and urea nitrogen compared with the cold season ( p < 0.01). Gut microbiota composition shifted seasonally, with Bacteroides dominating in warm seasons and Bacillus predominating in cold seasons. Functional metagenomics indicated cold-season enrichment in pathways related to carbon metabolism, ABC transporters, aminoacyl-tRNA biosynthesis, pyruvate metabolism, DNA replication, and methane metabolism ( p < 0.01). Metabolomics identified elevated warm-season microbial metabolites (His-Met, leucylleucine, luteolin 7-glucoside, ursolic acid; p < 0.05) and higher cold-season compounds (melatonin, glabrol, prostaglandin E2; p < 0.05), with KEGG enrichment linking these to steroid hormone biosynthesis, fatty acid metabolism, bile acid synthesis, and propanoate pathways. These findings suggest possible associations between seasonal extremes and: (1) modulation of nutrient metabolism (e.g., secondary bile acids and short-chain fatty acids); (2) activation of stress-response pathways (e.g., pentose phosphate pathway, ABC transporters, and DNA replication); and (3) immune regulation mediated by bioactive metabolites. Cold-season enrichment in DNA repair and energy-production pathways may be associated with responses to oxidative stress, whereas warm-season shifts in lipid metabolism are consistent with increased nutrient availability. Fluctuations in key metabolites—such as elevated melatonin in cold seasons and elevated ursolic acid in warm seasons—likely reflect adaptations related to thermoregulation and antioxidant defense. This work provides foundational insights into microbiota–host interactions under extreme environmental conditions, supporting the optimization of supplementation, probiotic use, and sustainable husbandry on the Qinghai–Tibet Plateau.

Qilian sheep are an important local breed of Tibetan sheep with strong adaptation to cold and hypoxic environments. To identify candidate genes related to nutrient metabolism in Qilian sheep, this study compared liver transcriptomes between Qilian sheep and Oula sheep raised under the same grazing and feeding conditions. Six 10-month-old ewes were selected from each breed, and three liver samples with high RNA quality from each group were used for transcriptome sequencing. Differential expression analysis identified 1640 differentially expressed genes under the thresholds of |log2FoldChange| > 1 and false discovery rate < 0.05, including 718 upregulated and 922 downregulated genes. KEGG enrichment analysis showed that these genes were mainly involved in lipid metabolism- and amino acid metabolism-related pathways, especially the peroxisome proliferator-activated receptor signaling pathway, fatty acid synthesis, and fatty acid beta-oxidation. qRT-PCR validation confirmed that RGN, LPGAT1, and BHMT2 were significantly upregulated, whereas SDS, GK, PC, MIOX, HMGCS2, PNPLA3, ACAA2, and HADHA were downregulated in Qilian sheep. These results indicate clear differences in liver nutrient metabolism-related gene expression between Qilian sheep and Oula sheep and provide a molecular basis for understanding the liver metabolic characteristics and adaptive metabolic mechanisms of Qilian sheep.