Dietary carbon loaded with nano-ZnO alters the gut microbiota community to mediate bile acid metabolism and potentiate intestinal immune function in fattening beef cattle.

BACKGROUND: To our knowledge, carbon loaded with nano-ZnO (NZnOC) represents a new nutritional additive for the animal husbandry industry. However, the mechanism by which NZnOC mediates beef cattle growth and intestinal health is not fully understood. This study aimed to investigate the effects of carbon loaded with nano-ZnO (NZnOC) supplementation on growth performance, gut microbiota, bile acid (BAs) metabolism and intestinal immunity in fattening cattle. Twenty cattle (16 ± 0.95 months) were randomly assigned to two dietary groups: CON (control, without feed additive) and NZnOC (diet supplemented with 80 mg NZnOC/kg diet dry matter basic) for 60 d. The colon digesta microbiota composition and BAs concentration were determined by microbiota metagenomics and gas chromatography methods, respectively. RESULTS: The results showed that the NZnOC-supplemented cattle had greater final weight, average daily gain and gain-to-feed ratio than those in the CON group. Cattle fed the NZnOC diet had a higher relative abundance of the secondary BAs synthesizing phyla Firmicutes, Tenericutes and Actinobacteria than those fed the CON diet. Dietary supplementation with NZnOC increased the relative abundance of the secondary BAs synthesis microbiota genera Clostridium, Ruminococcus, Eubacterium, and Brevibacillus in colon digesta. Cattle fed the NZnOC diet had increased activities of 3α-hydroxysteroid dehydrogenase (EC: 1.1.1.52) and bile acid-CoA ligase BaiB (EC: 6.2.1.7) in the colon digesta compared with those fed the CON diet. The primary BAs taurocholic acid, taurochenodeoxycholic acid and taurodeoxycholate acid were significantly decreased by dietary NZnOC supplementation, while the secondary BAs deoxycholic acid, taurolithocholic acid, beta-muricholic acid, 12-ketolithocholic acid and ursodeoxycholic acid were significantly increased. Dietary supplementation with NZnOC increased the mRNA abundance of G protein-coupled bile acid receptor 1, protein kinase cAMP-activated catalytic subunit alpha, cyclic-AMP response element binding protein 1 and interleukin (IL)-10 in the colon mucosa of cattle, while the mRNA abundance of tumor necrosis factor and IL-1ß were significantly decreased. CONCLUSIONS: In summary, dietary supplementation with NZnOC can facilitate the growth performance and intestinal immune function of cattle by improving BAs metabolism. NZnOC can be supplemented in the diet as a safe regulator of gut microbiota and as a feed additive in the ruminants industry.

Comparative transcriptome analysis of winter yaks in plateau and plain.

The yak is an important source for the people living and ecological environment in the Qinghai-Tibet Plateau. In every winter, many domestic yaks will lose bodyweight or dead under cold and food scarcity. Moving the plateau yaks to farm in the plain is a useful approach to reduce their environmental stress and gain more production. In this study, we measured growth, slaughter and beef quality traits every month and sequenced mRNA expression levels of muscles of two groups yaks living in plateau and plain respectively. We found there is significant difference (p-value <0.01) in the third (60 days), fourth (90 days), fifth (120 days) and sixth (150 days) weights between subpopulations in the plateau and plain. We identified 540 different expressed genes (DEGs) including 123 known genes and 417 unknown genes. Using the weighted correlation network analysis (WGCNA) to build a co-express network, the modules were strong relative to weight traits. The findings highlighted the underlying way and a relative network to yield a new view about gene expression between the yaks living plateau and plain.

Skeletal muscle proteome analysis provides insights on high altitude adaptation of yaks.

The differences in proteome profile of longissimus thoracis (LT) muscles of yak (Bos grunniens) and cattle (Bos taurus) were investigated employing isobaric tag for relative and absolute quantification (iTRAQ) approach to identify differentially expressed proteins and to understand the cellular level adaptations of yaks to high altitudes. Fifty-two proteins were differentially expressed in the two species, among which 20 were up-regulated and 32 were down-regulated in yaks. Gene ontology (GO) annotation revealed that most of the differentially expressed proteins were involved in the molecular function of protein binding, catalytic activity, and structural activity. Protein-protein interaction analysis recognized 24 proteins (involved in structural integrity, calcium ion regulation, and energy metabolism), as key nodes in biological interaction networks. These findings indicated that mammals living at high altitudes could possibly generate energy by pronounced protein catabolism and glycolysis compared with those living in the plains. The key differentially expressed proteins included calsequestrin 1, prostaglandin reductase 1 and ATP synthase subunit O, which were possibly associated with the cellular and biochemical adaptation of yaks to high altitude. These key proteins may be exploited as candidate proteins for mammalian adaptation to high altitudes.

Comparison of Y-chromosome-linked TSPY, TSPY2, and PRAMEY genes in Taurus cattle, yaks, and interspecific hybrid bulls.

Domestic yaks (Bos grunniens) and domestic Taurus cattle (Bos taurus) are closely related. An interesting phenomenon in interspecific crossings is male sterility in the F1 hybrid (yattle) and F2 backcross, with no late meiotic cells or spermatids in the seminiferous tubules. The mammalian Y chromosome is crucial for spermatogenesis and male fertility. This study investigated the copy number variations and mRNA of Y-transitional region genes TSPY2 (testis specific protein, Y-linked 2 and testis-specific Y-encoded protein 3-like) and PRAMEY (preferentially expressed antigen in melanoma, Y-linked), and Y-ampliconic region genes TSPY (testis-specific Y-encoded protein 1-like), ZNF280BY (zinc finger protein 280B, Y-linked) and HSFY (heat-shock transcription factor, Y-linked) in mature testes from Taurus cattle, yaks, and yattle. Phylogenetic trees divided 33 copies of TSPY into major 2 types (TSPY-T1 and TSPY-T2), 19 copies of TSPY2 into 2 types (TSPY2-T1 and T2), and 8 copies of PRAMEY into 4 types (PRAMEY-T1 to T4). Searching by the Basic Local Alignment Search Tool of the TSPY2 coding sequences in GenBank revealed that TSPY2 was conserved in Bovidae. The TSPY2-T2 sequences were absent, whereas PRAMEY-T2 and PRAMEY-T4 were amplified on the yak Y chromosome. The average copy numbers of TSPY-T2 and ZNF280BY were significantly different between cattle and yaks. The TSPY-T2, TSPY2, PRAMEY, ZNF280BY, and HSFY genes were uniquely or predominantly expressed in testes. Reverse-transcription quantitative PCR showed that the TSPY-T2, PRAMEY-T2, HSFY, ZNF280BY, protamine 1 (PRM1), and protamine 2 (PRM2) genes were almost not expressed in yattle. The PRM1 and PRM2 genes are used as positive markers for spermatozoa. Thus, our results showed that the genomic structure of the Y-transitional and Y-ampliconic region differed between Taurus cattle and yaks. Dysregulated expression of Y-ampliconic region genes TSPY-T2, HSPY, ZNF280BY, and Y-transitional region gene PRAMEY-T2 may be associated with hybrid male sterility in yattle.

Dynamic changes of yak (Bos grunniens) gut microbiota during growth revealed by polymerase chain reaction-denaturing gradient gel electrophoresis and metagenomics.

OBJECTIVE: To understand the dynamic structure, function, and influence on nutrient metabolism in hosts, it was crucial to assess the genetic potential of gut microbial community in yaks of different ages. METHODS: The denaturing gradient gel electrophoresis (DGGE) profiles and Illumina-based metagenomic sequencing on colon contents of 15 semi-domestic yaks were investigated. Unweighted pairwise grouping method with mathematical averages (UPGMA) clustering and principal component analysis (PCA) were used to analyze the DGGE fingerprint. The Illumina sequences were assembled, predicted to genes and functionally annotated, and then classified by querying protein sequences of the genes against the Kyoto encyclopedia of genes and genomes (KEGG) database. RESULTS: Metagenomic sequencing showed that more than 85% of ribosomal RNA (rRNA) gene sequences belonged to the phylum Firmicutes and Bacteroidetes, indicating that the family Ruminococcaceae (46.5%), Rikenellaceae (11.3%), Lachnospiraceae (10.0%), and Bacteroidaceae (6.3%) were dominant gut microbes. Over 50% of non-rRNA gene sequences represented the metabolic pathways of amino acids (14.4%), proteins (12.3%), sugars (11.9%), nucleotides (6.8%), lipids (1.7%), xenobiotics (1.4%), coenzymes, and vitamins (3.6%). Gene functional classification showed that most of enzyme-coding genes were related to cellulose digestion and amino acids metabolic pathways. CONCLUSION: Yaks' age had a substantial effect on gut microbial composition. Comparative metagenomics of gut microbiota in 0.5-, 1.5-, and 2.5-year-old yaks revealed that the abundance of the class Clostridia, Bacteroidia, and Lentisphaeria, as well as the phylum Firmicutes, Bacteroidetes, Lentisphaerae, Tenericutes, and Cyanobacteria, varied more greatly during yaks' growth, especially in young animals (0.5 and 1.5 years old). Gut microbes, including Bacteroides, Clostridium, and Lentisphaeria, make a contribution to the energy metabolism and synthesis of amino acid, which are essential to the normal growth of yaks.

Effects of Starvation on Lipid Metabolism and Gluconeogenesis in Yak.

This research was conducted to investigate the physiological consequences of undernourished yak. Twelve Maiwa yak (110.3±5.85 kg) were randomly divided into two groups (baseline and starvation group). The yak of baseline group were slaughtered at day 0, while the other group of yak were kept in shed without feed but allowed free access to water, salt and free movement for 9 days. Blood samples of the starvation group were collected on day 0, 1, 2, 3, 5, 7, 9 and the starved yak were slaughtered after the final blood sample collection. The liver and muscle glycogen of the starvation group decreased (p<0.01), and the lipid content also decreased while the content of moisture and ash increased (p<0.05) both in Longissimus dorsi and liver compared with the baseline group. The plasma insulin and glucose of the starved yak decreased at first and then kept stable but at a relatively lower level during the following days (p<0.01). On the contrary, the non-esterified fatty acids was increased (p<0.01). Beyond our expectation, the ketone bodies of ß-hydroxybutyric acid and acetoacetic acid decreased with prolonged starvation (p<0.01). Furthermore, the mRNA expression of lipogenetic enzyme fatty acid synthase and lipoprotein lipase in subcutaneous adipose tissue of starved yak were down-regulated (p<0.01), whereas the mRNA expression of lipolytic enzyme carnitine palmitoyltransferase-1 and hormone sensitive lipase were up-regulated (p<0.01) after 9 days of starvation. The phosphoenolpyruvate carboxykinase and pyruvate carboxylase, responsible for hepatic gluconeogenesis were up-regulated (p<0.01). It was concluded that yak derive energy by gluconeogenesis promotion and fat storage mobilization during starvation but without ketone body accumulation in the plasma.

Metagenomic Analysis Reveals A Gut Microbiota Structure and Function Alteration between Healthy and Diarrheic Juvenile Yaks.

Diarrhea-induced mortality among juvenile yaks is highly prevalent in the pastoral areas of the Qinghai-Tibet plateau. Although numerous diseases have been linked to the gut microbial community, little is known about how diarrhea affects the gut microbiota in yaks. In this work, we investigated and compared changes in the gut microbiota of juvenile yaks with diarrhea. The results demonstrated a considerable drop in the alpha diversity of the gut microbiota in diarrheic yaks, accompanied by Eysipelatoclostridium, Parabacteroides, and Escherichia-Shigella, which significantly increased during diarrhea. Furthermore, a PICRust analysis verified the elevation of the gut-microbial metabolic pathways in diarrhea groups, including glycine, serine, and threonine metabolism, alanine, aspartate, oxidative phosphorylation, glutamate metabolism, antibiotic biosynthesis, and secondary metabolite biosynthesis. Taken together, our study showed that the harmful bacteria increased, and beneficial bacteria decreased significantly in the gut microbiota of yaks with diarrhea. Moreover, these results also indicated that the dysbiosis of the gut microbiota may be a significant driving factor of diarrhea in yaks.

Whole-genome resequencing reveals genetic diversity, differentiation, and selection signatures of yak breeds/populations in southwestern China.

The Sichuan-Yunnan region is the main production area of yaks in southwestern China, with rich genetic resources of Yaks. Nevertheless, there have been limited study on the genetic characteristics of the entire yak populations in Tibet and southwestern China. In this study, we performed whole-genome resequencing to identify genetic variation information in a total of 198 individuals from six yak breeds (populations) in Sichuan (Muli yak, Jinchuan yak, Changtai yak, Maiwa yak), Yunnan (Zhongdian yak), and Tibet (Tibetan yak). The aim was to investigate the whole-genome genetic diversity, population genetic structure, and genome selection signatures. We observed that all six populations exhibit abundant genetic diversity. Except for Tibetan yaks, which showed low nucleotide diversity (0.00104), the remaining yak populations generally displayed high nucleotide diversity (0.00129-0.00153). Population genetic structure analysis revealed that, among the six yak populations, Muli yak exhibited greater differentiation from other yak populations and formed a distinct cluster independently. The Maiwa yak population displayed a complex genetic structure and exhibited gene exchange with Jinchuan and Changtai yaks. Positive selection signals were detected in candidate genes associated with growth (GNB4, HMGA2, TRPS1, and LTBP1), reproduction (PI4KB, DYNC1I1, and GRIP1), immunity (CD200 and IL1RAP), lactation (SNX13 and CPM), hypoxia adaptation (NDUFB6, PRKN, and MRPS9), hair (KRT24, KRT25, and KRT26), meat quality (SUCLG2), digestion and absorption (CLDN1), and pigment deposition (OCA2) using the integrated Pi and F ST methods. This study provides significant insights into understanding the whole-genome genetic characteristics of yak populations in Tibet and southwestern China.

Yak DEFB124 alleviates intestinal injury caused by Staphylococcus aureus infection.

To investigate the characteristics and functions of yak ß-defensin 124 (DEFB124), prokaryotic expression, analysis of gut microbiological and other methods were used in this study. The results showed that the sequence of yak DEFB124 gene was 306 bp in length and 207 bp in open reading frame, which encoded 68 amino acids. Yak DEFB124 protein was highly conserved and had the closest relationship with cattle. Yak DEFB124 protein was a secreted cationic ß-defensin. The recombinant expression plasmid pET32a-DEFB124 was constructed, and an about 24 kDa protein was successfully expressed. Yak DEFB124 protein had inhibitory activity against Staphylococcus aureus (S. aureus), and its MIC value was 64 µg/mL. After treating with yak DEFB124 protein, the activities of alkaline phosphatase (AKP) and total superoxide dismutase (T-SOD) were higher (P < 0.01) in the jejunum tissue, but the activity of lysozyme (LZM) was lower (P < 0.01). The number of goblet cells in the duodenum, jejunum, and ileum of the mice in the DEFB124 group was increased (P < 0.01). Besides, the expressions of MUC2 mRNA and protein were increased (P < 0.05) after the treatment with yak DEFB124 protein. Furthermore, the relative abundance of Lactobacillus in jejunum of mice in DEFB124 group was also increased. In summary, yak DEFB124 protein could increase the number of goblet cells in mice intestine and the abundance of intestinal probiotics Lactobacillus, thereby protecting the intestinal tract and alleviating intestinal damage.

Antimicrobial activity of yak beta-defensin 116 against Staphylococcus aureus and its role in gut homeostasis.

Staphylococcus aureus (S. aureus) is one of the most common food-borne poisoning microbial agent. However, the antimicrobial activity of ß-defensin 116 in yak and its application in S. aureus-induced diarrheal disease have not been reported. In this study, 303 bp cDNA sequence of yak DEFB116 gene was obtained. In addition, the prokaryotic expression vector of DEFB116 protein with a molecular weight of 16 kDa was successfully constructed and expressed. The yak DEFB116 gene can encode 19 amino acids, the percentage of hydrophobic amino acids is 36 % and the total positive charge is 6, which has potential antibacterial potential. Sufficient DEFB116 protein concentration and time can destroy the integrity of the bacterial cell membrane, resulting in leakage of intracellular solutes and thus killing S. aureus. The intestinal histopathological features and the number of inflammatory cells were improved in the diarrhea mouse model under the action of DEFB116 protein. The decrease of goblet cells was reversed, the expression of mucoprotein was increased. DEFB116 protein increased the abundance of Lactobacillus johnsonii, Lactobacillus reuteri and Desulfovibrio, and inhibited the reproduction of pathogenic bacteria. These findings provide new insights into the potential future applications of yak ß-defencins in the food industry and medical fields.

Yak-Derived CXCL14 Activates the Pro-Inflammatory Response of Macrophages and Inhibits the Proliferation and Migration of HepG2.

CXCL14 (C-X-C motif chemokine ligand 14) is an important chemokine involved in infection and immunity and plays an important role in a variety of immune-related diseases. The 446 bp cDNA sequence of the CXCL14 gene in yaks was obtained. Additionally, the prokaryotic expression vector of the CXCL14 protein with a molecular weight of 27 kDa was successfully constructed and expressed. The proliferation activities and migration abilities of spleen macrophages were significantly inhibited after treatment with the CXCL14 protein at different concentrations (1, 10 and 20 µg/mL) (p < 0.05). Furthermore, the expressions of pro-inflammatory cytokines interleukin 1 beta (IL-1ß), interleukin 6 (IL6), interleukin 8 (IL8) and interferon-α (TNF-α) were significantly increased (p < 0.05), but the expression of anti-inflammatory factor interleukin 10 (IL10) was significantly decreased (p < 0.05). The contents of inflammatory factors in the supernatant of cells were detected using ELISA, and it was also found that the contents of TNF-α, IL6 and cytochrome c oxidase subunit II (COX2) were significantly increased under different CXCL14 protein concentrations (p < 0.05). Finally, the exogenous addition of CXCL14 inhibited the activity, clonal formation and migration of hepatoma cells (HepG2). Additionally, after HepG2 cells were treated with 20 µg/mL CXCL14 protein for 12 h, 24 h and 36 h, the expression levels of BCL2 homologous antagonist/killer (BAK) and the BCL2-associated X apoptosis regulator (BAX) were increased to varying degrees, while the expression levels of hypoxia-inducible factor 1 subunit alpha (HIF1A), the mechanistic target of rapamycin kinase (mTOR) and cyclin-dependent kinase 1 (CDK1) genes decreased compared to the control group. In conclusion, the CXCL14 protein can inhibit the proliferation and migration of HepG2 cells by inducing the expression of macrophage pro-inflammatory factors and activating apoptosis-related genes to exert innate immunity. These results are helpful to further study the function of the CXCL14 protein and provide research data for the innate immune mechanism of yaks under harsh plateau environments.

Transcriptomic analysis reveals differentially expressed genes and key immune pathways in the spleen of the yak (Bos grunniens) at different growth stage.

Yak is one of the rare and unique cattle species on the Qinghai-Tibetan Plateau, which has strong adaptability to the extreme environment of the plateau. The spleens are important functional organs that enable animals to adapt to their external environment and are vital in the growth and development process. To further investigate changes in immune function during yak development, we compared the transcriptome profiles of spleen tissues among juvenile (1-day old), youth (15-months old), and prime (5-years old) yaks. Immunology of spleen development was evaluated based on histological analyses and global gene expression was examined by using RNA-sequencing (RNA-seq) technology. In this work, we found 6378 genes with significant differences between the spleen of juvenile yak and youth yak, with the largest difference between groups. There were 3144 genes with significant differences between the spleen of young yak and prime yak, with the smallest differences between groups. Further, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted for the functional annotation of these genes. GO and KEGG analysis showed that some of them were related to growth, disease, immune, and metabolism. However, the genetic mechanism underlying the adaptability of yak spleens at different ages to harsh plateau environments remains unknown. These findings are important for studying the mechanisms of spleen development in yaks of different age groups.

Comparative proteomic analysis of spleen reveals key immune-related proteins in the yak (Bos grunniens) at different growth stages.

Spleen plays an indispensable role in the immune system as the largest lymphatic organ in the body. The spleens of yaks at three developmental stages (1 day fetal yak, 15 months juvenile yak and 5 years old adult yak) were sampled and the Tandem mass tag (TMT) quantification method was employed in spleen proteomic analysis. The results showed that 6576 proteins and 529 differentially expressed proteins (DEPs) were identified in the yak spleens at three growth stages. Gene ontology (GO) analysis of DEPs indicated that DEPs were enriched in Oxygen transport, Actin filament movement, DNA replication, Cell cycle process, and Cell macromolecule biosynthesis process, which was conducive to high altitude breathing, protein synthesis and organ growth in yaks. These were indispensable for yak spleen growth and cell metabolism, high altitude adaptation. Those DEPs were further analyzed based on Kyoto encyclopedia of genes and genomes (KEGG) pathways, which principally participated in Th1 and Th2 cell differentiation, NF-kappa B signaling pathway, Phagosome, and Glutathione metabolism. Those pathways were associated with some animal life activities in defense against microbial antigens, indicating that with age, the immune function of the yak's spleen continued to increase. Hemoglobin, Tumor necrosis factor receptor associated factor 1 (TRAF1), T cell receptor (TCR), Macrophage receptor, Fc receptors (FcR), and Gamma-glutamyl transferase (GGT) of DEPs played roles in immune function in yak spleen directly or indirectly. The dynamic changes of Toll like receptor 2 (TLR2), TRAF1 and Heat shock protein 27 (HSP27 or HSPB1) detected by Immunohistochemistry were consistent with those obtained from TMT proteomic. In conclusion, this study provides extensive and functional analyses of the spleen proteome at three developmental stages and will offer a new insight into key proteins involved in the immune function of yak spleen.

Molecular characterization, expression and anti-tumor function analysis of yak IFITM2 gene.

IFITM2 is interferon-induced transmembrane protein 2, which plays an extremely key role in anti-tumor and anti-virus diseases. In this study, the 602 bp cDNA sequence of the yak (Bos grunniens) IFITM2 (BgIFITM2) gene was obtained. Moreover, the prokaryotic expression vector of BgIFITM2 protein was constructed and expressed successfully, with a molecular weight of 33.680 kDa. The proliferation activities and migration abilities of HepG2 cells were significantly inhibited after treatment with BgIFITM2 protein (0.1 and 1 µg/mL) (P < 0.05). The expressions of B cell lymphoma-2 (BCL2)/BCL2-associated X (BAX) and molecular target of rapamycin (mTOR) genes were significantly decreased, but the expressions of BAX gene were significantly increased after treatment with BgIFITM2 protein (0.1 and 1 µg/mL) (P < 0.05). The expression of BAX protein was also significantly increased after treatment with 1 µg/mL BgIFITM2 protein (P < 0.05). Finally, the addition of BgIFITM2 protein attenuated the formation of tumor lesions in mice, and the pathological damage of the lung was less than that in the model group. The expression of Ki67 protein in the model group was significantly higher than that in the control group (P < 0.05), but the expression of Ki67 protein in the BgIFITM2 group was significantly lower than that in the model group (P < 0.05). Taken together, BgIFITM2 protein could inhibit the proliferative activity of HepG2 cells by regulating apoptosis-related genes, and reduce the invasiveness of HepG2 cells in mice lung tissue. These results facilitate further studies on the function of BgIFITM2 protein.

Ruminal pH pattern, fermentation characteristics and related bacteria in response to dietary live yeast (Saccharomyces cerevisiae) supplementation in beef cattle.

OBJECTIVE: In this study we aimed to evaluate the effect of dietary live yeast supplementation on ruminal pH pattern, fermentation characteristics and associated bacteria in beef cattle. METHODS: This work comprised of in vitro and in vivo experiments. In vitro fermentation was conducted by incubating 0%, 0.05%, 0.075%, 0.1%, 0.125%, and 0.15% active dried yeast (Saccharomyces cerevisiae, ADY) with total mixed ration substrate to determine its dose effect. According to in vitro results, 0.1% ADY inclusion level was assigned in in vivo study for continuously monitoring ruminal fermentation characteristics and microbes. Six ruminally cannulated steers were randomly assigned to 2 treatments (Control and ADY supplementation) as two-period crossover design (30-day). Blood samples were harvested before-feeding and rumen fluid was sampled at 0, 3, 6, 9, and 12 h post-feeding on 30 d. RESULTS: After 24 h in vitro fermentation, pH and gas production were increased at 0.1% ADY where ammonia nitrogen and microbial crude protein also displayed lowest and peak values, respectively. Acetate, butyrate and total volatile fatty acids concentrations heightened with increasing ADY doses and plateaued at high levels, while acetate to propionate ratio was decreased accordingly. In in vivo study, ruminal pH was increased with ADY supplementation that also elevated acetate and propionate. Conversely, ADY reduced lactate level by dampening Streptococcus bovis and inducing greater Selenomonas ruminantium and Megasphaera elsdenii populations involved in lactate utilization. The serum urea nitrogen decreased, whereas glucose, albumin and total protein concentrations were increased with ADY supplementation. CONCLUSION: The results demonstrated dietary ADY improved ruminal fermentation dosedependently. The ruminal lactate reduction through modification of lactate metabolic bacteria could be an important reason for rumen pH stabilization induced by ADY. ADY supplementation offered a complementary probiotics strategy in improving gluconeogenesis and nitrogen metabolism of beef cattle, potentially resulted from optimized rumen pH and fermentation.

MicroRNAs miR-27a and miR-143 regulate porcine adipocyte lipid metabolism.

MicroRNAs (miRNAs) are non-coding small RNAs that play roles in regulating gene expression. Some miRNAs have been classed as epigenetic regulators of metabolism and energy homeostasis. Previous reports indicated that the miRNAs miR-27a and miR-143 were involved in lipid metabolism in human and rodents. To determine the roles of porcine miR-27a and miR-143 in adipocyte lipid metabolism, porcine adipocytes were cultured and allowed to induce differentiation for 10 days. The lipid-filled adipocytes were then transfected with miRNA mimics and inhibitors. We measured how the indicators of adipogenesis and adipolysis in porcine adipocytes were affected by the over-expression and by the inhibition of both miR-27a and miR-143. The results indicated that the over-expression of miR-27a could accelerate adipolysis releasing significantly more glycerol and free fatty acids than the negative control (P < 0.001), while the mimic of miR-143 expression, promoted adipogenesis by accumulating more triglycerides (P < 0.001) in the adipocytes. In addition, we demonstrated that there was good correlation (r > 0.98, P < 0.001) between the indicators of adipolysis in cell lysates and in the culture medium.

Promoter hypermethylation of PIWI/piRNA pathway genes associated with diminished pachytene piRNA production in bovine hybrid male sterility.

Hybrid male sterility (HMS) is a postzygotic reproductive isolation mechanism that enforces speciation. A bovine example of HMS is the yattle (also called dzo), an interspecies hybrid of taurine cattle (Bos taurus) and yak (Bos grunniens). The molecular mechanisms underlying HMS of yattle are not well understood. Epigenetic modifications of DNA methylation and P-element induced wimpy testis (PIWI)-interacting RNA (piRNAs) are important regulators in spermatogenesis. In this study, we investigated DNA methylation patterns and piRNA expression in adult testes in hybrid infertile yattle bulls and fertile cattle and yak bulls using whole genome bisulphite-seq and small RNA-seq. Promoter hypermethylation in yattle were associated with DNA methylation involved in gamete generation, piRNA metabolic processes, spermatogenesis, and spermatid development (P < 2.6 × 10-5). Male infertility in yattle was associated with the promoter hypermethylation-associated silencing of PIWI/piRNA pathway genes including PIWIL1, DDX4, PLD6, MAEL, FKBP6, TDRD1 and TDRD5. The downstream effects of silencing these genes were diminished production of 29- to 31- nucleotide pachytene piRNAs in yattle testes. Hypermethylation events at transposable element loci (LINEs, SINEs, and LTRs) were found in yattle. LINE-derived prepachytene piRNAs increased and SINE-derived prepachytene piRNAs were reduced in yattle testes. Our data suggests that DNA methylation affects the PIWI/piRNA pathway and is involved in gene expression and pachytene piRNA production during spermatogenesis in bovine HMS. DNA hypermethylation and disruption of piRNA production contributed to unsuccessful germ cell development that may drive bovine HMS.

Microbial diversity in the rumen, reticulum, omasum, and abomasum of yak on a rapid fattening regime in an agro-pastoral transition zone.

The ruminant digestive system harbors a complex gut microbiome, which is poorly understood in the case of the four stomach compartments of yak. High-throughput sequencing and quantitative PCR were used to analyse microbial communities in the rumen, reticulum, omasum, and abomasum of six domesticated yak. The diversity of prokaryotes was higher in reticulum and omasum than in rumen and abomasum. Bacteroidetes predominated in the four stomach compartments, with abundance gradually decreasing in the trend rumen > reticulum > omasum > abomasum. Microorganism composition was different among the four compartments, all of which contained high levels of bacteria, methanogens, protozoa and anaerobic fungi. Some prokaryotic genera were associated with volatile fatty acids and pH. This study provides the first insights into the microorganism composition of four stomach compartments in yak, and may provide a foundation for future studies in this area.

Comparative analysis of the microRNA transcriptome between yak and cattle provides insight into high-altitude adaptation.

Extensive and in-depth investigations of high-altitude adaptation have been carried out at the level of morphology, anatomy, physiology and genomics, but few investigations focused on the roles of microRNA (miRNA) in high-altitude adaptation. We examined the differences in the miRNA transcriptomes of two representative hypoxia-sensitive tissues (heart and lung) between yak and cattle, two closely related species that live in high and low altitudes, respectively. In this study, we identified a total of 808 mature miRNAs, which corresponded to 715 pre-miRNAs in the two species. The further analysis revealed that both tissues showed relatively high correlation coefficient between yak and cattle, but a greater differentiation was present in lung than heart between the two species. In addition, miRNAs with significantly differentiated patterns of expression in two tissues exhibited co-operation effect in high altitude adaptation based on miRNA family and cluster. Functional analysis revealed that differentially expressed miRNAs were enriched in hypoxia-related pathways, such as the HIF-1α signaling pathway, the insulin signaling pathway, the PI3K-Akt signaling pathway, nucleotide excision repair, cell cycle, apoptosis and fatty acid metabolism, which indicated the important roles of miRNAs in high altitude adaptation. These results suggested the diverse degrees of miRNA transcriptome variation in different tissues between yak and cattle, and suggested extensive roles of miRNAs in high altitude adaptation.

Comparative transcriptomics of 5 high-altitude vertebrates and their low-altitude relatives.

Background: Species living at high altitude are subject to strong selective pressures due to inhospitable environments (e.g., hypoxia, low temperature, high solar radiation, and lack of biological production), making these species valuable models for comparative analyses of local adaptation. Studies that have examined high-altitude adaptation have identified a vast array of rapidly evolving genes that characterize the dramatic phenotypic changes in high-altitude animals. However, how high-altitude environment shapes gene expression programs remains largely unknown. Findings: We generated a total of 910 Gb of high-quality RNA-seq data for 180 samples derived from 6 tissues of 5 agriculturally important high-altitude vertebrates (Tibetan chicken, Tibetan pig, Tibetan sheep, Tibetan goat, and yak) and their cross-fertile relatives living in geographically neighboring low-altitude regions. Of these, ∼75% reads could be aligned to their respective reference genomes, and on average ∼60% of annotated protein coding genes in each organism showed FPKM expression values greater than 0.5. We observed a general concordance in topological relationships between the nucleotide alignments and gene expression-based trees. Tissue and species accounted for markedly more variance than altitude based on either the expression or the alternative splicing patterns. Cross-species clustering analyses showed a tissue-dominated pattern of gene expression and a species-dominated pattern for alternative splicing. We also identified numerous differentially expressed genes that could potentially be involved in phenotypic divergence shaped by high-altitude adaptation. Conclusions: These data serve as a valuable resource for examining the convergence and divergence of gene expression changes between species as they adapt or acclimatize to high-altitude environments.