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Autophagy plays an important role in mammalian oocyte maturation and early embryonic development and rapamycin is well known for inducing autophagy. Although previous studies have reported the effects of rapamycin on oocytes in vitro maturation (IVM) in different species, few studies have been reported on the role of rapamycin in yak oocytes IVM and embryonic development. Therefore, the objective of this study was to examine the effect of rapamycin treatment on yak oocytes IVM and early embryonic development. Specifically, immature yak oocytes during IVM or parthenogenetic (PA) embryos were treated with different rapamycin concentrations to select an optimal dose. Then evaluated its effect on maturation rates, cleavage, and blastocyst formation rates, mitochondrial membrane potential, ROS levels. Related genes and proteins expression in matured oocytes and blastocysts were also evaluated. The results show that 10 nM rapamycin treatment during IVM significantly improved oocyte maturation rates of oocytes and blastocyst formation rates. Treatment with 10 nM rapamycin reduced ROS level but increased mitochondrial membrane potential. Correspondingly, mRNA and protein expressions of LC3, Beclin-1, and Bcl-2 up-regulated while Bax down-regulated in matured yak COCs. When parthenogenetic embryos were treated with different rapamycin concentrations, 10 nM rapamycin treatment showed higher 8-cell and blastocyst formation rates. Also, CDX2, POU5F1, SOX2, and Nanog levels in blastocysts were upregulated. In summary, our findings demonstrate that rapamycin treatment improves oocytes maturation probably by increasing mitochondrial membrane potential, reducing ROS levels, and regulating the apoptosis in mature yak oocytes. Rapamycin treatment also improves embryonic developmental competence in the yak.

We investigated the structural features of arterial blood vessels in yak uterine caruncle and the effects of the expression of vascular regulation-related factors on angiogenesis in pregnant and non-pregnant yak uterus. Three-dimensional specimens of the uterine artery of non-pregnant and pregnant yaks were produced to observe and measure the distribution characteristics and number of arterial vessels in the uterus and caruncle in the two periods. The uterine caruncle structure was observed and analysed by haematoxylin-eosin staining. The expression features of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in the uterine caruncle were detected with immunohistochemistry, quantitative real-time PCR (qRT-PCR) and western blotting. The length and number of blood vessels in the caruncle were increased, the degree of curvature was decreased, and the folding was more complicated during pregnancy as compared with that during non-pregnancy. The immunohistochemical results demonstrated that VEGF and Ang-1 were mainly expressed strongly in the mucosal epithelial cytoplasm. The glandular lumen of the uterine gland, lymphocytes and the media and adventitia of blood vessels are widely distributed, and they are all positive. VEGF and Ang-1 mRNA and protein levels were highest in pregnancy, followed by that in the luteal phase and in the follicular phase, and three stages were significantly different (p .05). These findings provide an anatomical reference and theoretical basis for improving the diagnosis and treatment of yak reproductive disorders and other diseases in high-altitude and low-oxygen environments.

The aim of this study was to investigate protein regulation at different time points during the in vitro maturation of yak oocytes. Yak oocytes at GV, MI, and MII stages were collected during in vitro maturation, and differential proteomics sequencing was performed using iTRAQ technology. GO functional classification indicated that the differential proteins were closely associated with biological processes such as “metabolic processes”, and molecular events such as “binding” molecular-function-related categories were active. KOG analysis showed that energy-metabolism-related activities were vigorous during oocyte development from the GV phase to MI phase, and genetic material preparation activities were more active when oocytes developed from the MI stage to MII stage. KEGG pathway analysis showed that the PPAR metabolic pathway, Hippo signaling pathway, and ECM–receptor interaction and metabolic pathway were enriched from the GV to the MI stages. The PI3K-Akt, TGF-β, and phagosome pathways were enriched from the MI stage to the MII stage. These results indicate that transient dynamic changes occurred in the proteome during the maturation of yak oocytes, and the physiological functions mediated by these were also different. The accurate identification of the differential proteins in the three stages of GV, MI, and MII was helpful in further analyzing the molecular regulatory mechanism of yak oocyte maturation.

The thymus is the site of development and maturation of functional T lymphocytes and is critically important to the immune system. The purpose of this study was to examine the expression of markers of T lymphocytes, macrophages, dendritic cells, B lymphocytes and plasmocytes in the yak thymus.Twenty healthy male yaks were divided into newborn (2-4 weeks old, n = 10) and adult (3-4 years old, n = 10) group. qRT-PCR was used to evaluate the mRNA expression level of the main markers of the studied cell types. Immunohistochemistry was used to detect the distribution of CD3+ T lymphocytes, CD68+ macrophages, SIRPα+ dendritic cells, CD79α+ B lymphocytes, IgA and IgG+ plasmocytes.Within the same age group, the mRNA expression of CD3ε was highest (P0.05), followed by that of CD68, SIRPα, CD79α, IgG and IgA. Furthermore, CD3ε, CD68, and SIRPα mRNA expression levels were higher in newborn yaks than in the adult ones (P0.05), whereas those of CD79α, IgA, and IgG were higher in adults (P0.05). Immunohistochemical results showed localization of CD3+ T lymphocytes in the thymic cortex and medulla. CD68+ macrophages, SIRPα+ dendritic cells, CD79α+ B lymphocytes, IgA+ and IgG+ plasmocytes were mainly observed in the cortico-medullary region and medulla. In the same age group, the frequency of CD3+ T lymphocytes was higher than that of CD68+ macrophages and SIRPα+ dendritic cells (P0.05), followed by those of CD79α+ B lymphocytes and IgA+ and IgG+ plasmocytes. No significant difference was observed between B lymphocyte and plasmocyte frequencies in the yak thymus in both age groups (P0.05). The frequency of CD3+, CD68+ and SIRPα+ cells decreased from newborns to adults (P0.05). However, the frequencies of CD79α+, IgA+ and IgG+ cells increased from newborn to adult yaks (P0.05).The thymus of newborn yaks is well-developed, with higher numbers of T lymphocytes, macrophages, and dendritic cells than those in the adult thymus. However, higher frequencies of plasmocytes and B lymphocytes were detected in the adult thymus, suggesting that adults may better resist infections through humoral immunity as this organ undergoes involution. Furthermore, there was no significant difference in the number of IgA and IgG plasmocytes, which differs from what is observed in rodents and humans. This difference might be related to the fact that yaks live in low-oxygen plateaus.

The gas-phase environment of in vitro culture system plays an important role in the development of oocytes, and oxygen concentration is one of the important factors. In the present study, we aimed to explore the effect of different oxygen concentrations (20%, 10%, 5% or 1% O

High-altitude adaptation is viewed as a biologically based process during which yaks respond to the harsh condition of coldness and low oxygen. Although high-altitude adaptation has been studied intensively, there is a lack of multi-omics analysis on its physiological mechanism. Therefore, in this study, 10X Genomics single-cell technology were adopted to identify cellular heterogeneity and trajectory differentiation in different parts of the yak oviduct, thus further exploring new cell markers and ligand-receptors. Besides, based on the comparison of oviduct epithelial cells from the ampulla and isthmus between the yak and bovine, genes and pathways related to altitude adaptation were discussed by bulk-seq. Single cell RNA sequencing (scRNA-seq) revealed 14 cell types defined by multiple specific molecular markers, the main cell types are epithelial cells, ciliated cells, secretory cells, basal cells, mesenchymal stem cells, T cells, B cells, monocytes, NK cells, macrophages, and fibroblasts, among which basal cells could be differentiated into secretory cells and ciliated cells. Apart from that, immune cells were the apex products of cell development, while ciliated cells had the characteristics of de-differentiation, and the new ligand-receptors CD74-COPa and CD74-APP were found. In addition, by bulk-seq, it was found that in the ampulla, there were 5149 differential genes (1875 up-regulated genes and 3274 down-regulated genes). YME1L1 was down-regulated significantly, whereas PPIB, PCBD, CYTB, RPL24, RPS5, and UBL5 were up-regulated to a great extent. In the isthmus, there were 6313 differential genes (2146 up-regulated genes and 4167 down-regulated genes). Among them, YME1L1, GSTA4, DNTTIP2, TMCO6, LRBA, and MYH14 were down-regulated obviously, while SNRPD3, CYTB, ADIPOQ, RNH1, PRDX5, DPF1, CRYAB, and PRXL2B were up-regulated dramatically. Through the comparison, it was observed that GSTA4 and YME1L1 were down-regulated, while CYTB was up-regulated in the ampulla and the isthmus. Beyond that, the oxidative phosphorylation pathway was explored. In conclusion, it was demonstrated that ciliated cells were featured with dedifferentiation, and new ligand receptors CD74-COPa and CD74-APP were discovered. Furthermore, GSTA4, YME1L1 and CYTB were main molecules of altitude adaptation, and the oxidative phosphorylation pathway was the key pathway of high-altitude adaptation. This study provides basic data on the heterogeneity and cell trajectory of yak oviduct from the single-cell sequence, and provides the key genes and pathways of yak high-altitude adaptation, as well as the theoretical basis and the data for revealing the low pregnancy rate and genetic breeding of yaks.

The goal of this study was to characterize and evaluate the main markers of macrophages, T lymphocytes, B lymphocytes and plasmocytes in the testis of juvenile and adult yaks by quantitative real-time polymerase chain reaction and immunohistochemistry. Within the same age group, the mRNA expression of CD68 was always highest, followed by that of CD3ε, CD79α, IgG and IgA. Moreover, CD68, CD3, CD79α, IgA and IgG positive cells were all located in the testicular interstitial tissues of juvenile and adult yaks. In the same age group, the frequency of CD68 positive macrophages was higher than that of CD3 positive T lymphocytes, which was followed by that of CD79α positive B lymphocytes and IgA and IgG positive plasmocytes. No significant difference was observed between the B lymphocyte and plasmocyte frequencies in yak testes. Furthermore, CD68, CD3ε, CD79α, IgA and IgG mRNA expression levels and the frequencies of CD68, CD3, CD79α, IgA and IgG positive cells increased from juveniles to adults. Similarly, the frequencies of CD68, CD3, CD79α, IgA and IgG positive cells also increased with age. These results suggest that in the yak testis, the immune defence system against pathogens might primarily comprise macrophages and T lymphocytes in the testicular interstitial tissue. Moreover, the testicular immune environment may mature and expand to a fully functional state in adult yaks. The low frequencies of B lymphocyte and plasmocyte in yaks, differing from those in rodents and humans, might be related to the fact that yaks live in low-oxygen plateaus.

The potential reproduction power of domestic animals is limited by a complicated follicular atresia process. P53, caspase-9 (Casp9), Bax, Bcl-2 and Fas play a crucial role in the ovarian mitochondrion-dependent apoptosis and death receptor pathway. In accordance with this study, the expression levels of Casp9, Bax, Bcl-2 and Fas were analysed in ovaries and oviducts of yak by immunohistochemistry (IHC). P53 and the above in ovarian granulosa cells (GCs) from atretic (3-6 mm) to healthy follicles (6-8 mm) and in oviducts were examined from the luteal phase to the follicular phase during the oestrous circle by Western blot (WB) and real-time PCR (RT-PCR). Results demonstrated that typical classic apoptotic factors Casp9, Bax, Bcl-2 and Fas were expressed in the cytoplasm and zonal pellucida of oocytes, primordial follicles, primary follicles, ovarian surface epithelium, ovarian GCs, granular lutein cells, surface epithelia in oviduct uterotubal junction and oviduct ampulla during the luteal phase. RT-PCR and WB revealed that P53 and Fas significantly increased in GCs of atretic follicles. P53 and Casp9 increased in oviduct epithelium during the luteal phase, but Fas was unchanged. A contrary tendency was noted in Bcl-2 and Bax expression. Overall, P53 and Fas play an essential role in inducing GC apoptosis, and Bax, Bcl-2, Casp9 and P53 are involved in oviduct epithelial regeneration in yak.

Mammalian oocyte maturation and early embryo development are highly sensitive to the in vitro culture environment, and oxygen concentration is one of the important factors. In the present study, we aimed to explore the effects of different oxygen concentrations (20%, 10%, 5% or 1% O2) on yak oocyte maturation, in vitro fertilization (IVF), and embryo development competence, as well as its effects on the oxidative response, metabolism, and apoptosis in cumulus-oocyte complexes (COCs) and the embryo. The results revealed that the maturation rate of oocytes, blastocysts rate and hatched blastocysts rate in the group with 5% oxygen concentration were significantly higher (P

The yak (Bos grunniens) is the most important livestock animal in high-altitude regions owing to its prominent adaptability to cold conditions, nutritional deficiencies and hypoxia. The reproductive organs exhibit different histological appearances and physiological processes at different reproductive stages. Hypoxia-inducible factor-1 alpha (HIF-1α) is the regulatory subunit of HIF-1 that crucially regulates the response to hypoxia in mammalian organisms. The goal of our study was to investigate the expression and distribution of HIF-1α in the primary yak reproductive organs at different reproductive stages. Samples of the ovary, oviduct and uterus of 15 adult female yaks were collected and used in the experiment. The expression and localization of HIF-1α proteins and mRNA were investigated using quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB) and immunohistochemistry (IHC). The results indicated that the expression of HIF-1α protein in the ovary was higher during the luteal phase than during the follicular phase and gestation period (p < .05). In the oviduct, HIF-1α protein was also more highly expressed during the luteal phase than during the follicular phase and gestation period (p < .01). However, in the uterus, the HIF-1α protein had stronger expression during the gestation period than during the follicular phase (p < .01) and luteal phase (p < .05). The expression of HIF-1α mRNA was similar to that of its protein. Immunohistochemical analysis revealed intense immunostaining of HIF-1α proteins in the follicular granulosa cells, granular luteal cells, villous epithelial cells of the oviduct, endometrial glandular epithelium and luminal epithelium, foetal villous trophoblast, and epithelia of caruncular crypts. This study showed that the expression of HIF-1α in the ovary, oviduct and uterus varies according to the stage of the reproductive cycle. This implies that HIF-1α plays an important role in regulating the stage-specific physiological function of yak reproductive organs under hypoxic environments.

High-quality oocytes are a prerequisite for successful fertilization. Mammals feeding on aflatoxin-contaminated feed can cause reproductive toxicity, including follicular atresia, poor oocyte development and maturation, and aberrant epigenetic modifications of oocytes. In addition, the important role of ascorbic acid (AA) in reproductive biology has been confirmed, and AA is widely used as an antioxidant in cell culture. However, the toxic effects of aflatoxin B

Background and Materials and methods: In order to clarify and reveal the morphological characteristics of yak placentomes, placentomes obtained from 151 to 180 days of pregnant yaks were observed using light microscopy and scanning and transmission electron microscopy. Results: The results indicated that sessile, dome-shaped yak placentomes seemed to have a relatively complex villous-crypt architecture pattern. There was a straight maternal plate beneath the placentome. Plentiful uterine glands and a dense cellular layer were present in the endometrium lamina propria close to the maternal plate. Trophoblast giant cells appeared to have similar ultrastructure features to these in other ruminants, including abundant mitochondria, an extensive array of rough endoplasmic reticulum, advanced Golgi complex and many specific secretory granules. Trophoblast giant cells could also secrete neutral and acid glycoconjugates. Furthermore, numerous glycoconjugates were distributed in the connective zones between mononuclear trophoblast cells and crypt epithelial cells as well as in maternal connective tissues. Mononucleate trophoblast cells, which had abundant microvilli that interdigitated with the corresponding microvilli arising from the crypt epithelial cells, had numerous mitochondria and vesicles, but there were no glycoconjugates. Conclusions: The morphological structures of yak placentomes were similar to those of other bovid genera; however, certain differences were observed. These findings might provide morphological evidence for evolutionary relationships between different bovid genera.

Background Porcine circovirus type 3 (PCV3) has been confirmed to infect pigs, posing a health risk and making pigs more susceptible to other pathogens. After the first report of PCV3 infection in the United States, its prevalence was determined in pigs suffering from clinical digestive or respiratory diseases in several other regions, including the Sichuan and Gansu provinces of China. In this study, we describe the frequency of PCV3 detection in Tibetan pigs inhabiting three different provinces surrounding the Qinghai-Tibet Plateau of China. Methods A total of 316 samples from diarrheic animals and 182 samples from healthy animals were collected in a randomized manner. Conventional PCR was applied for PCV3 DNA detection. The conserved regions of the PCV3 gene were analyzed with MEGA 7.1 software to design specific primers to sequence entire Cap genes in PCV3 strains, and the sequences were then used to confirm the subtypes of PCV3 in the positive samples. Prediction of the amino acid sequences by nucleotide sequence translation was also performed to compare the point mutations in the entire Cap protein. Twenty PCV3 whole-genomic sequences were used for genome phylogenetic analyses of PCV3 and sequence alignments with 22 other reference strains. Results We found that the prevalence of the virus was significantly higher in samples from pigs with diarrhoea than that in samples from healthy pigs. Phylogenetic analysis of Cap proteins demonstrated that the 20 PCV3 strains formed three clades, including PCV3a (8/20, 40.00%), PCV3b (5/20, 25%) and PCV3c (7/20, 35.00%). The complete genome sequence revealed that these strains formed one branch in the phylogenetic tree. Sequence analysis showed that the Cap proteins of the 20 different viral strains shared between 95.84 and 99.18% nucleotide identity. Cap protein sequence analyses showed that the positivity rate of PCV3a was highest in the samples from pigs with diarrhoea. In comparison, PCV3c was the most elevated subtype in the healthy samples. There was no mutation at a specific site in the amino acid sequences of the entire Cap protein from different PCV3 subtype strains from heathy samples. There was a mutation at site 113 in PCV3a, site 129 in PCV3b, and site 116 in PCV3c. Conclusion Our present data provide evidence that PCV3 is prevalent in Tibetan pigs at high altitudes in China, and the higher prevalence rates of the PCV3a and PCV3b subtypes in samples from pigs with diarrhoea further indicate that the genotypes should not be neglected during surveys of the pathogenicity of PCV3. Phylogenetic and genetic diversity analyses suggested that the continuous evolution, adaptation and mechanisms of pathogenicity of PCV3 in Tibetan pigs living in this special environment should be further studied.

Autophagy is one of the basic cellular mechanism during preimplantation development of mammalian embryos, and it plays crucial role in several physiological processes. It is induced by interleukin (IL)-1β in mammalian cells. Our present study shows that IL-1β is important for autophagy activation in embryo development. Our in vitro culture system analysis shows effect of IL-1β in medium on the development of mouse embryos and it was found to be concentration dependent. A preimplantation embryo culture using medium containing IL-1β did not improve cleavage and blastocyst development rates of mouse embryos; however, blastocyst quality was significantly improved by increasing total cell number, especially in supplementary 20 ng/mL IL-1β. Furthermore, autophagy activation mainly occurs in 2 to 4 cell embryo and blastocyst, 20 ng/mL IL-1β into culture medium can effectively enhance levels of messenger RNA and protein of autophagy-related-factors in 2 to 4 cell embryos and blastocyst, while these factors reduce in VGX-1027 (IL-1β inhibitor) groups that also reduce the quality of blastocyst. Effects of IL-1β on the development of embryo reduced in 20 ng/mL IL-1β supplemented group when 5 mM 3-methyladenine (3-MA) was also added, which used to inhibit autophagy activation in endogenous PtdIns3Ks signal pathway. Our current results show that exogenous IL-1β can effectively induce autophagy in mouse embryos at stages of 2 to 8 cell and blastocyst, that also help to improve the quality of blastocyst.

DNA methylation as an important, essential epigenetic modification is critical for the successful development of mammalian embryos. In recent years, the important role of ascorbic acid (AA) as an irreplaceable cofactor for epigenetic regulation has been confirmed. However, the effect of AA on DNA methylation in preimplantation embryo development of plateau yak remains unknown. In this study, we explored whether AA can help regulates DNA methylation in yak preimplantation embryos to improve the blastocyst quality. First, our results indicate that the preimplantation of the yak still follows the classical pattern of DNA demethylation and remethylation, however, remethylation occurs in the blastocyst stage. Second, the unique expression pattern of the ten-eleven translocation enzyme (TET3) in the cytoplasm plays a key role in the demethylation mechanism. Third, in the blastocyst stage, the pluripotency gene CDX2 promoter region was in a hypomethylated state, and the POU5F1, SOX2, and NANOG promoter regions were in moderate methylation states. In addition, treatment with 50 μg/ml AA mainly improved the expression levels of DNMT1, DNMT3a, and TET3, ensured the establishment, maintenance and transition of 5-methylcytosine. After AA treatment, the methylation level of the pluripotency genes NANOG promoter regions was significantly reduced, and the mRNA transcript abundance of the pluripotency genes NANOG, POU5F1, and CDX2 was upregulated. In conclusion, our findings suggest that AA could increase blastocyst cell numbers by regulating DNA methylation of yak preimplantation embryos .

Tetraparvovirus, formerly known as Partetravirus, is a newly discovered genus in the family Parvoviridae that is considered phylogenetically distinct from other parvoviruses. However, nothing is known about the prevalence of Tetraparvovirus in special livestock living on the Qinghai-Tibet Plateau of China, such as Tibetan pigs and Tibetan sheep. A pair of special primers was designed based on the conserved regions in the genome of ungulate tetraparvovirus 2 (P-PARV4) and ungulate tetraparvovirus 4 (O-PARV4) and was used to detect P-PARV4 in domestic pigs and Tibetan pigs and O-PARV4 in ovines and Tibetan sheep. The results showed a 15.59 and 9.38% prevalence of P-PARV4 in domestic pigs (18.96% in Gansu Province and 11.76% in Qinghai Province) and Tibetan pigs (14.28% in Gansu Province and 4.44% in Qinghai Province), respectively, and a 7.31 and 5.20% prevalence of O-PARV4 in ovines (6.61% in Gansu Province and 8.00% in Qinghai Province) and Tibetan sheep (4.55% in Gansu Province and 5.50% in Qinghai Province), respectively. The prevalence of P-PARV4 was 14.76% (31/210) for ≤1-month-old pigs and 10.58% (20/189) for > 1-month-old pigs, and the positive rates of O-PARV4 were 7.65% (18/235) for ≤1-month-old sheep and 5.05% (11/218) for > 1-month-old sheep. The phylogenetic analysis of NS1, VP1, VP2 and the whole PARV4-related provirus genome demonstrated that both P-PARV4 and O-PARV4 sequences in this study were more closely related to the sequences of other strains discovered in the same genus of animals. The identity analyses for the full-length VP2 genomes of O-PARV4 revealed 98.84–100.00% sequence identity among the 7 strains and the previously reported strain, which was 98.60–99.28% for P-PARV4. In the present study, for the first time, we have provided comprehensive information regarding the widespread infection of P-PARV4 and O-PARV4 in special livestock on the Qinghai-Tibet Plateau in China. Our present findings highlight the importance of epidemiologic surveillance to limit the spread of Tetraparvovirus in livestock at high altitudes in China. Electronic supplementary material The online version of this article (10.1186/s12985-019-1167-z) contains supplementary material, which is available to authorized users.

Transforming growth factor-β (TGF-β) and heat shock protein 70 (HSP70) are important for the hair follicle (HF) cycle, but it is unclear whether they participate in HF regression in yak skin. In this study, we investigated the role of TGF-β, TGF-βRII, and HSP70 in the transition from anagen to catagen of HFs. The results showed that TGF-β2 transcription was significantly higher than that of TGF-β1 and TGF-β3 in the same periods. Meanwhile, the expressions of TGF-β2, TGF-βRII, and caspase-3 were higher in the catagen phase than that in mid-anagen, and some TGF-βRII-positive HF cells were terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive. Moreover, the HSP70 protein levels in mid-anagen were higher than those in late-anagen and catagen. These results suggested that TGF-β2 plays a major role in catagen induction in yak HFs, which might be achieved via TGF-βRII-mediated apoptosis in HF epithelial cells. In contrast, HSP70 might protect epithelial cells from apoptosis and ultimately inhibit HF regression. In conclusion, TGF-β2 has positive effects, whereas HSP70 has negative effects, on catagen induction.

Bovine viral diarrhea virus (BVDV) is an enveloped virus with an RNA genome, causing serious economic losses to the areas dominated by livestock industry. Currently, although several compounds with biological activities of inhibiting virus replication have been reported, amino acid mutations (especially F224S mutation) frequently occurring in the RNA-dependent RNA polymerase (RdRp) have greatly reduce their value of further research. In this study, we introduced an effective and rapid in silico strategy to explore the differences in the binding modes of VP32947 between the wild/mutant-type RdRp at the molecular level, and further explained the main reasons for the variations in the inhibitory activities of VP32947 against the two types of enzymes. Firstly, the binding site of VP32947 in the finger domain was determined based on the previously reported experimental data, and the initial conformation of VP32947 in the wild RdRp was constructed using molecular docking. Then, the mutant research system was obtained directly by artificial mutation strategy. Afterwards, the built research systems were subjected to microsecond-timescale molecular dynamic simulation, and the conformational and energic profile analyses were performed according to the simulation trajectories. It was found that after 1 μs simulation, VP32947 in the mutant system was transferred to the left side of Loop α, and its interactions with the residues in the loop region were weakened. However, VP32947 in the wild system remained at the right side of Loop α, and could have a good fit with the sub-pocket formed by F224, I261, P262, N264, S532, which was conducive to maintaining its stable binding conformation in the wild RdRp. The illustration of the difference in the binding mechanisms of VP32947 in the wild/mutant RdRp would provide a theoretical basis for the rational design of innovative inhibitors based on the enzyme.

To evaluate age-related changes in the morphology as well as the expression and localization of IgA and IgG in yak pharyngeal tonsils, 20 healthy yaks were divided into four age groups [newborn (1-7 days old), juvenile (5-7 months old), adult (3-6 years old) and old (7-10 years old)]. Morphologic characteristics were observed by histological techniques. The expression and localization of IgA and IgG in pharyngeal tonsils were detected by enzyme linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. The results showed that the epithelium of the pharyngeal tonsils included nonreticular epithelium with an intact basement membrane and reticular epithelium with a discontinuous basement membrane and nonepithelial cell infiltration. In newborn yaks, only primary lymphoid follicles were observed in pharyngeal tonsils. In other age groups, both primary and secondary lymphoid follicles were observed, but some of the lymphoid follicles in the old yaks were degenerated. The number of lymphoid follicles increased from the newborn to the adult group and peaked in the adult group, but the number decreased in the old group. In addition, the age-related trends of IgA and IgG protein expression were similar to those of the number of lymphoid follicles. The concentration of IgG was significantly higher than that of IgA in all age groups. Both IgA and IgG antibody secreting cells (ASCs) were distributed in the subepithelial region of the nonreticular epithelium, the reticular epithelium, the lymphoid follicles, the interfollicular areas and in between the salivary glands. The densities of IgA and IgG ASCs in pharyngeal tonsils were similar to the expression trend of both proteins in each age group. The results indicate that the morphology and amount of lymphoid follicles in yak pharyngeal tonsils vary with age. Pharyngeal tonsils produce more IgG than IgA, indicating that IgG could be significant component of mucosal immune responses in yaks.

Hypoxia-inducible factor-1α (HIF-1α) and heme oxygenase-1 (HO-1) are important transcription regulators in hypoxic cells and for maintaining cellular homeostasis, but it is unclear whether they participate in hypoxia-induced excessive proliferation of yak pulmonary artery smooth muscle cells (PASMCs). In this study, we identified distribution of HIF-1α and HO-1 in yak lungs. Immunohistochemistry and immunofluorescence results revealed that both HIF-1α and HO-1 were mainly concentrated in the medial layer of small pulmonary arteries. Furthermore, under induced-hypoxic conditions, we investigated HIF-1α and HO-1 protein expression and studied their potential involvement in yak PASMCs proliferation and apoptosis. Western blot results also showed that both factors significantly increased in age-dependent manner and upregulated in hypoxic PASMCs (which exhibited obvious proliferation and anti-apoptosis phenomena). HIF-1α up-regulation by DMOG increased the proliferation and anti-apoptosis of PASMCs, while HIF-1α down-regulation by LW6 decreased proliferation and promoted apoptosis. More so, treatment with ZnPP under hypoxic conditions down-regulated HO-1 expression, stimulated proliferation, and resisted apoptosis in yak PASMCs. Taken together, our study demonstrated that both HIF-1α and HO-1 participated in PASMCs proliferation and apoptosis, suggesting that HO-1 is important for inhibition of yak PASMCs proliferation while HIF-1α promoted hypoxia-induced yak PASMCs proliferation.

Methylenetetrahydrofolate reductase (MTHFR), an enzyme expressed in mammalian testes, exerts a direct effect on spermatogenesis, however, its protein characteristics in bovine testes remain unknown. Here, we analysed bovine testicular structure, MTHFR bioinformatics profile, mRNA, and protein expression characteristics in yellow-cattle (y-c) and yak testis using histological procedures, bioinformatics analysis, qRT-PCR, and western blot. Testes from 13 bovines, &le, 2 years juvenile (y-c, n = 3, yak, n = 3) and &ge, 3 years adult (y-c, n = 3, yak, n = 4) were collected and analysed. Anatomical characteristics of testis in y-c and yak were similar except the weight or size for which that of y-c was significantly higher or greater than yak. In y-c, an open reading frame (ORF) for 2600 nucleotides sequence, encoding 655 amino acids showed high homology with zebu cattle (99.51%) and wild yak (98.68%). Secondary and 3D protein structures were similar to that of humans with differences in the number of nucleotides, amino acids, and some physico-chemical characteristics. MTHFR mRNA expression in y-c and yak were significantly higher in adult testes compared with juvenile ones. However, its protein expression was higher, but not statistically significant, in adult y-c and yak compared to the juvenile ones. The highlights and inferences of these and other findings are discussed.

A multiplex PCR method was developed to detect the main pathogens of Qinghai Tibetan sheep endometritis. First, the genomes of five standard bacterial strains were extracted and specific primers were selected; the multiplex PCR method was established by using the genome of the standard strain as a template. The samples were collected by sterile cotton swab from Tibetan sheep uterus, and then placed in LB medium and numbered. After 48 h, the genomes of cultured bacteria were extracted and detected by single PCR method, then the positive samples were recorded. The positive samples detected by single PCR were selected for multiplex PCR detection and recorded again. The coincidence rate between these two methods was calculated to measure the accuracy of multiplex PCR. In order to identify the species of the pathogen, 30 positive samples verified by single and multiplex PCR were randomly selected for bacterial isolation and identification. In the 600 samples, the infected ratio of Streptococcus agalactiae (GBS) was 47.33%, Escherichia coli 34.83%, Staphylococcus aureus 6.5%, Salmonella and Trueperella pyogenes were negatively detected. Among the positive samples detected by multiplex PCR, the positive ratio of GBS was 45.50%, E. coli 33.50%, S. aureus 6.5%. Comparison of two detection results, Multiplex PCR detection coincidence rate is more than 95%. The isolated pathogens were identified as E. coli, GBS and S. aureus, which was consistent with the results of two methods. The multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS, E. coli and S. aureus.本研究旨在建立一种多重PCR 方法检测青海藏绵羊子宫内膜炎主要的病原菌。首先，提取5 种标准菌株基因组，筛选出特异性引物；然后以标准菌株的基因组为模板，建立多重PCR 方法。用无菌棉拭子涂抹藏绵羊子宫，置于LB 培养液中培养并编号，48 h 后提取样品基因组。运用单一PCR 法对600 份样品基因组进行检测，记录阳性样品；再挑取单一PCR 法检测的阳性样品进行多重PCR 检测，再次记录阳性样品，通过计算两种检测方法的符合率验证多重PCR 方法；随机挑出30 份阳性样品，进行病原菌分离鉴定菌种种类。单一PCR 检测的样品中，无乳链球菌感染比例占47.33%，大肠杆菌占34.83%，金黄色葡萄球菌占6.5%，未检出沙门氏菌和化脓隐秘杆菌；多重PCR 检测的阳性样品中，无乳链球菌感染比例占45.50%，大肠杆菌占33.50%，金黄色葡萄球菌占6.5%；两种检测结果相比较，多重PCR 检测出的符合率均高于95%；分离鉴定的病原菌与两种PCR 方法检测出的菌种结果基本一致。成功建立了多重PCR 方法并检测出引起青海藏绵羊子宫内膜炎的主要病原菌为无乳链球菌、大肠杆菌和金黄色葡萄球菌。.

Endometritis is one of the main diseases which harm sheep husbandry. Astragalin and chlorogenic acid (CGA) are common active ingredients of traditional Chinese medicine (TCM) with immunoprotective, antioxidant, and anti-inflammatory properties. In the present study, sheep endometrial epithelium cells (SEECs) were successfully purified and identified, and the in vitro inflammation model of SEECs induced by Escherichia coli (E. coli) was successfully established. To explore the effect of astragalin and CGA on the inflammation induced by E. coli and its potential mechanism, six groups were set up, namely, group C, M, astragalin, CGA, BAY, and STR. Cells in group C were incubated with DMEM/F12 for 6 h, while cells in group M, astragalin, CGA, BAY, and STR were incubated with DMEM/F12, astragalin, CGA, BAY, and STR for 3 h, respectively, followed by E. coli infection at a multiplicity of infection (MOI) of 1 E. coli per cell for 3 h. Subsequently, the cells and the supernatant were collected to detect the expression of genes in the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway by ELISA, qPCR, and western blot. The results showed that E. coli could induce inflammation of SEECs in vitro, while astragalin and CGA could alleviate the inflammatory response induced by E. coli via inhibiting the activation of the TLR4/NF-κB signaling pathway, which provides a theoretical and experimental foundation for preventing sheep endometritis clinically.

The main reproductive organs undergo different histological appearances and physiological processes under different reproductive statuses. The variation of these organs depends on a delicate regulation of cell proliferation, differentiation, and apoptosis. Extracellular signal-regulated kinases1/2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) super family. They have important roles in regulating various biological processes of different cells, tissues, and organ types. Activated ERK1/2 generally promotes cell survival, but under certain conditions, ERK1/2 also have the function of inducing apoptosis. It is widely believed that ERK1/2 play a significant role in regulating the reproductive processes of mammals. The goal of our research is to investigate the expression and distribution of ERK1/2 in the yak&rsquo, s main reproductive organs during different stages. In the present study, samples of the ovary, oviduct, and uterus of 15 adult female yak were collected and used in the experiment. The ERK1/2 proteins, localization, and quantitative expression of their mRNA were investigated using immunohistochemistry (IHC), western blot (WB) and relative quantitative real-time polymerase chain reaction (RT-PCR). The results indicated that ERK1/2 proteins and their mRNA were highly expressed in the ovary of the luteal phase and gestation period, in the oviduct of the luteal phase, and in the uterus of the luteal phase and gestation period. Immunohistochemical analysis revealed a strong distribution of ERK1/2 proteins in follicular granulosa cells, granular luteal cells, villous epithelial cells of the oviduct, endometrial glandular epithelium, and luminal epithelium. These results demonstrated that the expression of ERK1 and ERK2 proteins and their mRNA in the yak&rsquo, s ovary, oviduct, and uterus varies with the stage of the reproductive cycle. The variation character of ERK1 and ERK 2 expression in the yak&rsquo, s main reproductive organs during different stages implies that they play an important role in regulating the reproductive function under different physiological statuses.