The dietary crude protein level could affect ruminal fermentation parameters and the microflora of ruminants. The present study\'s aim was to investigate the effects of different protein level diets on ruminal morphology, fermentation parameters, digestive enzyme activity, microflora and metabolites of Tibetan sheep. Ninety weaned lambs (initial weight of 15.40 ± 0.81 kg, 2 months old) were selected and randomly divided into three groups (six pens/treatment, five rams/pen). Dietary treatments were formulated with 13.03% (high protein, HP), 11.58% (moderate protein, MP) and 10.20% (low protein, LP), respectively. Compared with LP, both papillae length and papillae width were significantly promoted in HP and MP (p < 0.05). The concentrations of ammonia nitrogen, total VFAs, propionic acids and butyric acids in HP were significantly increased compared to those in MP and LP (p < 0.05). The activities of protease and α-amylase in HP were significantly greater than those of LP (p < 0.05). For the ruminal microbial community, higher proportions of phylum Prevotella 1 and Succiniclasticum and genus Rikenellaceae RC9 gut group and Ruminococcus 1 were observed in HP (p < 0.05). A total of 60 differential metabolites (DMs) (28 up, 32 down) between HP and MP; 73 DMs (55 up, 18 down) between HP and LP; and 65 DMs (49 up, 16 down) between MP and LP were identified. Furthermore, four pathways of the biosynthesis of unsaturated fatty acids, tryptophan metabolism, bile secretion and ABC transporters were significantly different (p < 0.05). The abundance of phylum Prevotella 1 was negatively associated with stearic acid and palmitic acid but positively associated with the taurine. The abundance of genus Ruminococcus 1 was negatively associated with stearic acid, oleic acid, erucic acid, Indole-3-acetamide and palmitic acid but positively associated with 6-hydroxymelatonin. In conclusion, a 13.03% CP level improved ruminal morphology, fermentation parameters and digestive enzyme activities through modulating the microbial community and regulating metabolism in Tibetan sheep.

UNASSIGNED: T-sheep and H-sheep exhibit different environmental adaptability and production performance. The rumen microbiome has co-evolved with hosts and plays a vital role in nutrient digestion and energy metabolism. In our previous study, we found that T-sheep have a higher efficiency in energy metabolism than H-sheep, but the rumen microbial community remains unclear.
UNASSIGNED: In this study, we determined the rumen bacterial profile and rumen fermentation parameters to reveal the bacterial profiles and predictive functions among breeds and diets with four different energy levels, as well as the correlation between bacterial profiles and rumen fermentation characteristics.
UNASSIGNED: The results showed that the rumen total volatile fatty acids (VFAs), acetate, butyrate, total branched-chain VFAs, iso-butyrate, and iso-valerate were higher in T-sheep than H-sheep. The alpha diversity of ruminal bacteria is not affected by dietary energy, but it shows a distinction between the sheep breeds. Specifically, T-sheep rumen bacteria exhibit higher alpha diversity than H-sheep. The beta diversity of ruminal bacteria is not influenced by dietary energy or sheep breeds, indicating similar communities of ruminal bacteria between different diets and sheep breeds. The phyla of Bacteroidetes and Firmicutes predominate in the rumen, with a higher relative abundance of Firmicutes observed in T-sheep than H-sheep. The two most abundant genera in the rumen were Prevotella 1 and Rikenellaceae RC9 gut group. Prevotella 1 is the predominant bacterial genus in the rumen of H-sheep, while the Rikenellaceae RC9 gut group dominates in the rumen of T-sheep. Microbial co-occurrence network analysis reveals that variations in rumen fermentation characteristics result from differences in module abundance, with a higher abundance of VFA-producing modules observed in the rumen of T-sheep. Microbial function prediction analysis showed that dietary energy rarely alters the functional composition of rumen bacteria. However, there were differences in the functions of rumen bacteria between sheep breeds, with T-sheep showing a greater emphasis on energy metabolism-related functions, while H-sheep showed a greater emphasis on protein metabolism-related functions.
UNASSIGNED: These findings provide evidence of the special rumen microbial community that helps T-sheep efficiently obtain energy from low-protein and low-energy diets, enabling them to survive in the extreme environment of the Qinghai-Tibet Plateau.

BACKGROUND: Elucidating the genetic variation underlying phenotypic diversity will facilitate improving production performance in livestock species. The Tibetan sheep breed in China holds significant historical importance, serving as a fundamental pillar of Qinghai\'s animal husbandry sector. The Plateau-type Tibetan sheep, comprising 90% of the province\'s population, are characterized by their tall stature and serve as the primary breed among Tibetan sheep. In contrast, Zhashijia sheep exhibit larger size and superior meat quality. These two species provide an excellent model for elucidating the genetic basis of body size variation. Therefore, this study aims to conduct a comprehensive genome-wide association study on these two Tibetan sheep breeds to identify single nucleotide polymorphism loci and regulatory genes that influence body size traits in Tibetan sheep.
RESULTS: In this study, the phenotypic traits of body weight, body length, body height, chest circumference, chest depth, chest width, waist angle width, and pipe circumference were evaluated in two Tibetan sheep breeds: Plateau-type sheep and Zhashijia Tibetan sheep. Whole genome sequencing generated 48,215,130 high-quality SNPs for genome-wide association study. Four methods were applied and identified 623 SNPs significantly associated with body size traits. The significantly associated single nucleotide polymorphisms identified in this study are located near or within 111 candidate genes. These genes exhibit enrichment in the cAMP and Rap1 signaling pathways, significantly affecting animal growth, and body size. Specifically, the following genes were associated: ASAP1, CDK6, FRYL, NAV2, PTPRM, GPC6, PTPRG, KANK1, NTRK2 and ADCY8.
CONCLUSIONS: By genome-wide association study, we identified 16 SNPs and 10 candidate genes associated with body size traits in Tibetan sheep, which hold potential for application in genomic selection breeding programs in sheep. Identifying these candidate genes will establish a solid foundation for applying molecular marker-assisted selection in sheep breeding and improve our understanding of body size control in farmed animals.

Tibetan sheep are vital to the ecosystem and livelihood of the Tibetan Plateau; however, traditional breeding methods limit their production and growth. Modern molecular breeding techniques are required to improve these traits. This study identified a single nucleotide polymorphism (SNP) in myostatin (MSTN) and Callipyge in Tibetan sheep. The findings indicated notable associations between MSTN genotypes and growth traits including birth weight (BW), body length (BL), chest width (ChW), and chest circumference (ChC), as well as a particularly strong association with cannon circumference (CaC) at 2 months of age. Conversely, Callipyge polymorphisms did not have a significant impact on Tibetan sheep. Moreover, the analyses revealed a significant association between sex and BW or hip width (HW) at 2 months of age and ChW, ChC, and CaC at 4 months of age. Furthermore, the study\'s results suggested that the genotype of MSTN as a GA was associated with a notable sex effect on BW, while the genotype of Callipyge (CC) showed a significant impact of sex on CaC at 2 months of age. These results indicated that the SNP of MSTN could potentially serve as a molecular marker for early growth traits in Tibetan sheep.

Substituting traditional protein feed with palm kernel meal (PKM) in the diet of Tibetan sheep can be a cost-effective feeding strategy. To determine the impact of PKM on flavor development in different adipose tissues of Tibetan sheep, subjects were fed with 15% and 18% of PKM, while the control group received no PKM. The fatty acids and volatile compounds in the samples were then analyzed by GC-MS and HS-GC-IMS. Adding PKM to the diet significantly increased the C12:0, C14:0, C16:0 and C18:1N9 content in adipose tissues compared with the control, and most of these were associated with flavor formation (p < 0.05). The flavor compounds in the adipose tissues predominantly consisted of alcohols, ketones, acids and aldehydes. In particular, including PKM in the diet increased the proportion of ketones but decreased the proportion of alcohols, acids and aldehydes in subcutaneous and tail fat. Specifically, the proportion of acetone, acetoin monomer, 2,3-butanedione, 2-butanone monomer, 2-methyl-2-propanol, 2-methyl-2-propanol and methyl acetate increased significantly in the subcutaneous and tail fat (p < 0.05), while that of ethanol, 1-propanol monomer, butanol monomer, acetic acid monomer and acetic acid monomer decreased. Intermuscular fat exhibited variable results, mainly because the addition of PKM resulted in higher proportions of alcohols, including ethanol, 1-propanol and butanol monomer, especially at 15% PKM. In summary, the addition of PKM improved the flavor of Tibetan sheep fat and increased the amount of favorable volatile flavor compounds. This study can serve as reference for understanding the effects of dietary PKM on the adipose tissue flavor profile of Tibetan sheep.

BACKGROUND: Palm kernel meal (PKM) is a by-product of oil palm kernel after oil extraction, which is widely used in animal feeds due to its high energy content. This study aimed to investigate the impact of supplementing Tibetan sheep with PKM on their hepatic phenotype, oxidative stress and immune response. A total of 120 Tibetan lambs (Initial weight = 12.37 ± 0.92 kg) were randomly assigned into four groups: control group (C group, 0% PKM diet), low group (L group, 15% PKM diet), middle group (M group, 18% PKM diet), and high group (H group, 21% PKM diet) on a dry matter basis. The feeding experiment was performed for 130 d, including a 10 d adaption period.
RESULTS: Results showed that the level of GSH-Px were higher in the H and M groups than in the C and L groups (P < 0.05). The levels of IgM and TNF-α were higher in the M group when compared to those on the C group (P < 0.05). The level of IgA was significantly higher in the M group than in the H group (P < 0.05). Additionally, compared with the others groups, the hepatocytes in the M group displayed a radial arrangement, forming hepatic plates that were centered around the central vein. The transcriptome results revealed that proteasome 26 S subunit, ATPase 3 (PSMC3), proteasome 26 S subunit, ATPase 5 (PSMC5), proteasome 26 S subunit ubiquitin receptor, non-ATPase 4 (PSMD4), proteasome activator subunit 1 (PSME1), acyl-CoA dehydrogenase short/branched chain (ACADSB), enoyl-CoA hydratase, short chain 1 (ECHS1), serine dehydratase (SDS), ornithine transcarbamylase (OTC), and phenylalanine hydroxylase (PAH) were the hub genes regulating the amino acid metabolism in the liver.
CONCLUSIONS: In summary, dietary 18% PMK supplementation contributed to improve the hepatic phenotype, oxidative stress and immune response through regulating the expression of related genes.

The Baihe River, a tributary of the Yellow River located in the Ngawa Tibetan and Qiang Autonomous Prefecture in Northern Sichuan, is surrounded by natural resources suitable for animal development. However, the impact of livestock activities water microbiome in this area remains unexplored. This study collected water samples from areas with captive yaks and sheep (NS and YS) and compared them with water samples from Hongyuan Baihe River. Through amplicon sequencing, we investigated the impact of livestock activities on aquatic microorganisms. Diversity analysis, significance analysis, and microbial phenotype prediction indicated a significant decrease in microbial community diversity and function in the NS and YS groups. Pathogenic microorganisms such as Bacteroidales and Thelebolaceae and antibiotic-resistant bacteria genes such as Flavobacteriales and Burkholderiaceae were significantly higher in livestock breeding areas. Additionally, bacteria adapted to acidification, hypoxia, and eutrophication (e.g., Acidobacteria, Flavobacteriales, Deltaproteobacteria, Rhodobacterales) were more abundant in these areas. Our results demonstrate that livestock activities significantly alter the structure and function of microbial communities in surrounding water bodies, deteriorating water quality.

During the adaptive evolution of animals, the host and its gut microbiota co-adapt to different elevations. Currently, there are few reports on the rumen microbiota-hepato-intestinal axis of Tibetan sheep at different altitudes. Therefore, the purpose of this study was to explore the regulatory effect of rumen microorganism-volatile fatty acids (VFAs)-VFAs transporter gene interactions on the key enzymes and genes related to gluconeogenesis in Tibetan sheep. The rumen fermentation parameters, rumen microbial densities, liver gluconeogenesis activity and related genes were determined and analyzed using gas chromatography, RT-qPCR and other research methods. Correlation analysis revealed a reciprocal relationship among rumen microflora-VFAs-hepatic gluconeogenesis in Tibetan sheep at different altitudes. Among the microbiota, Ruminococcus flavefaciens (R. flavefaciens), Ruminococcus albus (R. albus), Fibrobactersuccinogenes and Ruminobacter amylophilus (R. amylophilus) were significantly correlated with propionic acid (p < 0.05), while propionic acid was significantly correlated with the transport genes monocarboxylate transporter 4 (MCT4) and anion exchanger 2 (AE2) (p < 0.05). Propionic acid was significantly correlated with key enzymes such as pyruvate carboxylase, phosphoenolpyruvic acid carboxylase and glucose (Glu) in the gluconeogenesis pathway (p < 0.05). Additionally, the expressions of these genes were significantly correlated with those of the related genes, namely, forkhead box protein O1 (FOXO1) and mitochondrial phosphoenolpyruvate carboxykinase 2 (PCK2) (p < 0.05). The results showed that rumen microbiota densities differed at different altitudes, and the metabolically produced VFA contents differed, which led to adaptive changes in the key enzyme activities of gluconeogenesis and the expressions of related genes.

Tibetan sheep were introduced to the Qinghai Tibet plateau roughly 3,000 B.P., making this species a good model for investigating genetic mechanisms of high-altitude adaptation over a relatively short timescale. Here, we characterize genomic structural variants (SVs) that distinguish Tibetan sheep from closely related, low-altitude Hu sheep, and we examine associated changes in tissue-specific gene expression. We document differentiation between the two sheep breeds in frequencies of SVs associated with genes involved in cardiac function and circulation. In Tibetan sheep, we identified high-frequency SVs in a total of 462 genes, including EPAS1, PAPSS2, and PTPRD. Single-cell RNA-Seq data and luciferase reporter assays revealed that the SVs had cis-acting effects on the expression levels of these three genes in specific tissues and cell types. In Tibetan sheep, we identified a high-frequency chromosomal inversion that exhibited modified chromatin architectures relative to the noninverted allele that predominates in Hu sheep. The inversion harbors several genes with altered expression patterns related to heart protection, brown adipocyte proliferation, angiogenesis, and DNA repair. These findings indicate that SVs represent an important source of genetic variation in gene expression and may have contributed to high-altitude adaptation in Tibetan sheep.

The aim of this study was to investigate the effects of ensiled agricultural byproducts from Qinghai-Tibet plateau on growth performance, rumen microbiota, ruminal epithelium morphology, and nutrient transport-related gene expression in Tibetan sheep. Fourteen male Tibetan sheep were randomly assigned to one of two diets: an untreated diet (without silage inoculum, CON, n = 7) or an ensiled diet (with silage inoculum, ESD, n = 7). The total experimental period lasted for 84 d, including early 14 d as adaption period and remaining 70 d for data collection. The ESD increased average daily gain (P = 0.046), dry matter intake (P < 0.001), ammonia nitrogen (P = 0.045), microbial crude protein (P = 0.034), and total volatile fatty acids concentration (P < 0.001), and decreased ruminal pH value (P = 0.014). The proportion of propionate (P = 0.006) and the copy numbers of bacteria (P = 0.01) and protozoa (P = 0.002) were higher, while the proportion of acetate (P = 0.028) was lower in the sheep fed ESD compared to CON. Pyrosequencing of the 16S ribosomal RNA gene revealed that ESD increased the relative abundance of Firmicutes, Ruminococcus, Lachnospiraceae_AC2044_group, Lachnospiraceae_XPB1014_group, and Christensenellaceae_R-7_group in the rumen (P < 0.05), while decreased the relative abundance of Bacteroidota, Prevotellaceae_UCG-003, and Veillonellaceae_UCG-001 (P < 0.05). Analyses with PICRUSt2 and STAMP indicated that the propionate metabolism pathway was enriched in the sheep fed ESD (P = 0.026). The ESD increased the rumen papillae height (P = 0.012), density (P = 0.036), and surface area (P = 0.001), and improved the thickness of the total epithelia (P = 0.018), stratum corneum (P = 0.040), stratum granulosum (P = 0.042), and stratum spinosum and basale (P = 0.004). The relative mRNA expression of cyclin-dependent Kinase 2, CyclinA2, CyclinD2, zonula occludens-1, Occludin, monocarboxylate transporter isoform 1 (MCT1), MCT4, sodium/potassium pump, and sodium/hydrogen antiporter 3 were higher in the rumen epithelial of sheep fed ESD than CON (P < 0.05). Conversely, the relative mRNA expressions of Caspase 3 and B-cell lymphoma-2 were lower in the sheep fed ESD than CON (P < 0.05). In conclusion, compared with an untreated diet, feeding an ensiled diet altered the rumen microbial community, enhanced nutrient transport through rumen epithelium, and improved the growth performance of Tibetan sheep.
Tibetan sheep on the Qinghai-Tibet Plateau experience significant nutrient stress while a substantial amount of agricultural byproducts in the region go discarded and wasted. In this study, agricultural byproducts were ensiled and fed to the Tibetan sheep to investigate their effects on growth performance, rumen microorganisms, and nutrient transport through rumen epithelial tissues. Fourteen male Tibetan sheep were randomly assigned to one of two diets: untreated diet (without silage inoculum, CON, n = 7) or ensiled diet (with silage inoculum, ESD, n = 7). After 70 d of feeding, the ESD-fed sheep had a higher body weight than CON. The ensiled diet changed the rumen microbial community and increased the relative abundance of cellulolytic bacteria in the rumen. In addition, the ensiled diet also promoted the development of rumen epithelia and improved the relative expression of gene related to nutrient transport. Overall, the ensiled diet optimized the use of agricultural byproducts and significantly contributed to the production of Tibetan sheep.