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Abstract Background Foot-and-mouth disease (FMD) is a devastating disease affecting cloven-hoofed animals, that leads to significant economic losses in affected countries and regions. Currently, there is an evident inclination towards the utilization of nanoparticles as powerful platforms for innovative vaccine development. Therefore, this study developed a ferritin-based nanoparticle (FNP) vaccine that displays a neutralizing epitope of foot-and-mouth disease virus (FMDV) VP1 (aa 140–158) on the surface of FNP, and evaluated the immunogenicity and protective efficacy of these FNPs in mouse and guinea pig models to provide a strategy for developing potential FMD vaccines. Results This study expressed the recombinant proteins Hpf, HPF-NE and HPF-T34E via an E. coli expression system. The results showed that the recombinant proteins Hpf, Hpf-NE and Hpf-T34E could be effectively assembled into nanoparticles. Subsequently, we evaluated the immunogenicity of the Hpf, Hpf-NE and Hpf-T34E proteins in mice, as well as the immunogenicity and protectiveness of the Hpf-T34E protein in guinea pigs. The results of the mouse experiment showed that the immune efficacy in the Hpf-T34E group was greater than the Hpf-NE group. The results from guinea pigs immunized with Hpf-T34E showed that the immune efficacy was largely consistent with the immunogenicity of the FMD inactivated vaccine (IV) and could confer partial protection against FMDV challenge in guinea pigs. Conclusions The Hpf-T34E nanoparticles stand out as a superior choice for a subunit vaccine candidate against FMD, offering effective protection in FMDV-infected model animals. FNP-based vaccines exhibit excellent safety and immunogenicity, thus representing a promising strategy for the continued development of highly efficient and safe FMD vaccines.

Abstract Understanding the microflora inhabiting the reproductive tract is important for a better understanding of female physiology and reproductive health. The endometrial fluid from mice in three reproductive stages (A: Unproductive mice; B: Postovulatory mice; C: Postpartum mice) was extracted for microbial DNA extraction and sequencing. Phenotypic and functional analyses of endometrial microbial enrichment was undertaken using LefSe. The results showed 95 genera and 134 species of microorganisms in the uteri of mice. There were differentially distributed genera, among which Lactobacillus, Enterococcus, and Streptococcus were more abundant in the endometrial fluid of mice in the unproductive group. That of mice in the postovulatory group was colonized with Salmonella enterica and Campylobacter and was mainly enriched in metabolic pathways and steroid biosynthesis. The presence of Chlamydia, Enterococcus, Pseudomonadales, Acinetobacter, and Clostridium in the endometrial fluid of postpartum mice, in addition to the enrichment of the endocrine system and the Apelin and FoxO signaling pathways, resulted in a higher number of pathogenic pathways than in the other two groups. The results showed that the microbial diversity characteristics in the endometrium of mice in different reproductive states differed and that they could be involved in the regulation of animal reproduction through metabolic pathways and steroid biosynthesis, suggesting that reproductive diseases induced by microbial diversity alterations in the regulation of animal reproduction cannot be ignored.

In animal assisted reproductive technology, the production of high-quality oocytes is crucial. The yak, having lived in the Qinghai-Tibet Plateau for an extended period, has reproductive cells that are regulated by hypoxia-inducible factor 1α (HIF-1α). This study aimed to investigate the impact of HIF-1α on yak oocyte maturation and early embryonic development in vitro through the regulation of autophagy. The in vitro maturation process of yak oocytes involved the addition of the HIF-1α inducer DFOM and the inhibitor LW6 to examine their effects on yak oocyte maturation, early embryonic development, cell autophagy, cytochrome P450s (CYP450s) enzyme expression, and cumulus diffusion factors. The findings revealed that DFOM significantly upregulated the expression of HIF-1α, resulting in increased the cumulus diffusion area, elevated first polar body expulsion rate of oocytes, enhanced mitochondrial and actin levels, decreased ROS production, and reduced early apoptosis levels of oocytes. Moreover, DFOM promoted the expression of autophagy-related proteins, CYP450s enzymes, and cumulus diffusion factors, thereby enhancing oocyte maturation and early embryonic development. Conversely, LW6 exhibited opposite effects. The inhibition of autophagy levels with 3-MA during DFOM treatment yielded similar outcomes. Furthermore, reducing autophagy led to increased apoptosis levels at all stages of early embryonic development, as well as a significant decrease in total cell number and ICM/TE ratio of blastocysts. Studies have shown that during the in vitro maturation of yak oocytes, HIF-1α can affect the cumulus expansion area of oocytes by regulating autophagy, the first polar body excretion rate, mitochondrial level, actin level, ROS and early apoptosis level, the CYP450s enzyme, and the expression of cumulus expansion factors, thereby improving the in vitro maturation and early embryonic development of yak oocytes. These findings offer valuable insights into the reproductive regulation mechanism of yaks in hypoxic environments and suggest potential strategies for the advancement of yak assisted reproductive technology.

To adapt to the extreme conditions of plateau environments, yaks have evolved thick hair, making them an ideal model for investigating the mechanisms involved in hair growth. We can gain valuable insights into how hair follicles develop and their cyclic growth in challenging environments by studying yaks. However, the lack of essential data on yak hair follicle histology and the absence of in vitro cell models for hair follicles serve as a limitation to such research objectives. In this study, we investigated the structure of skin tissue during different hair follicle cycles using the yak model. Additionally, we successfully established in vitro models of hair follicle-associated cells derived from yak skin, including dermal papilla cells (DPCs), preadipocytes, and fibroblasts. We optimized the microdissection technique for DPCs culture by simplifying the procedure and reducing the time required. Furthermore, we improved the methodology used to differentiate yak preadipocytes into mature adipocytes, thus increasing the differentiation efficiency. The introduction of yak as a natural model provides valuable research resources for exploring the mechanisms of hair growth and contributes to a deeper understanding of hair follicle biology and the development of regenerative medicine strategies.

Abstract Background Growing oocytes acquire the ability to mature through two-way communication between gametes and surrounding somatic cumulus cells (CCs). Granulosa cells (GCs) support oocyte growth, regulate meiosis progression, and modulate global oocyte transcription activity. However, the proliferation and differentiation of the yak ovary in GCs and CCs remain unclear. To characterize the important roles of long non-coding RNA, (lncRNA), microRNA (miRNA), and messenger RNA (mRNA), whole-transcriptome analysis was performed. Real-time quantitative fluorescence PCR was performed to verify the selected RNA sequences. Results Important gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways related to differentiation and oocyte development were identified for the target genes of differentially expressed lncRNAs, miRNAs, and mRNAs. In total,6223 mRNAs (2197 upregulated, 4026 downregulated), 643 lncRNAs (204 upregulated, 479 downregulated), and 559 miRNAs (311 upregulated, 248 downregulated) were significantly altered between the two groups. Target genes involved in cell adhesion, cell differentiation, regulation of developmental processes, cell proliferation, embryo development, signal transduction, apoptosis, and aromatic compound biosynthetic processes were significantly enriched. These RNAs were involved in ECM-receptor interaction, MAPK signaling, Hippo signaling, PI3K-Akt signaling, cell cycle, cell adhesion, leukocyte trans-endothelial migration, and actin cytoskeleton regulation. Conclusions A comprehensive analysis of the co-expression network of competing endogenous RNAs (ceRNAs) will facilitate the understanding of the process of granulosa cell proliferation and differentiation and offer a theoretical basis for the development of oocytes.

The yak is a unique creature that thrives in low-oxygen environments, showcasing its adaptability to high-altitude settings with limited oxygen availability due to its unique respiratory system. However, the impact of hypoxia on alveolar type II (AT2) epithelial cell proliferation in yaks remains unexplored. In this study, we investigated the effects of different altitudes on 6-month-old yaks and found an increase in alveolar septa thickness and AT2 cell count in a high-altitude environment characterized by hypoxia. This was accompanied by elevated levels of hypoxia-inducible factor-1α (HIF-1α) and epidermal growth factor receptor (EGFR) expression. Additionally, we observed a significant rise in Ki67-positive cells and apoptotic lung epithelial cells among yaks inhabiting higher altitudes. Our in vitro experiments demonstrated that exposure to hypoxia activated HIF-1α, EGF, and EGFR expression leading to increased proliferation rates among yak AT2 cells. Under normal oxygen conditions, activation of HIF-1α enhanced EGF/EGFR expressions which subsequently stimulated AT2 cell proliferation. Furthermore, activation of EGFR expression under normoxic conditions further promoted AT2 cell proliferation while simultaneously suppressing apoptosis. Conversely, inhibition of EGFR expression under hypoxic conditions had contrasting effects. In summary, hypoxia triggers the proliferation of yak AT2 cells via activation facilitated by the HIF-1α/EGF/EGFR signaling cascade.

Abstract 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.

IntroductionAutophagy and endocytosis are crucial biological activities in mammalian follicle development and oocyte maturation, which are easily affected by external environmental factors. Epidermal growth factor (EGF), as an important component of follicular fluid, regulates the growth and apoptosis of follicular cells. However, its regulatory mechanism of autophagy and endocytosis in mammals, especially in large domestic animals such as plateau yak, remains unclear. This study aimed to investigate the regulatory mechanism of EGF on autophagy and endocytosis in yak cumulus cells.MethodsYak cumulus cells were treated with different concentrations of EGF and appropriate concentrations of EGFR inhibitor gefitinib (10 μM). The dynamic expression levels of Atg5, Beclin1, LC3, Cav1 and Cav2 were detected by immunofluorescence staining, qRT-PCR and Western-blot.ResultsEGF inhibited autophagy in yak cumulus cells by down-regulating the expression of Atg5, Beclin1, and LC3. The level of autophagy varied with the concentration of ligands, and the inhibition was most significant at 100 ng/mL. Noteworthy, EGF can promote endocytosis by regulating the expression of Cav1 and Cav2, but the EGFR-mediated signaling pathway is not the main way to regulate the expression of these proteins.DiscussionThese results provide a reference for further exploring the effects of growth factors on livestock germ cells and the regulatory role of autophagy-endocytosis crosstalk mechanism in follicle development and oocyte maturation, to improve the fecundity of yaks.

The oviduct consists of three parts: the infundibulum (In), ampulla (Am), and isthmus (Is). These have the same histological structure, but different physiological functions. In this study, transcriptomics was used to analyze mRNA in these three parts of yak oviduct. The results showed that there were 325 up-regulated genes and 282 down-regulated genes in the infundibulum and ampulla. Moreover, there were 234 up-regulated genes and 776 down-regulated genes in the isthmus and ampulla, as well as 873 up-regulated genes and 297 down-regulated genes in the infundibulum and isthmus. The expression of C3 in the infundibulum was significantly higher than that in the ampulla and isthmus. The expression of FAU in the isthmus was significantly lower than that in the ampulla and infundibulum, and the expression of EEF1A1 in the ampulla was significantly higher than that in the ampulla and infundibulum. When the infundibulum was compared with the ampulla and isthmus, it was found that the up-regulated genes were enriched in the lysosome, phagosome, staphylococcus aureus infection, and leishmaniasis pathway. When the isthmus was compared with the ampulla and infundibulum, the up-regulated genes were present in the apoptosis pathway, oxidative phosphorylation, and viral myocarditis pathway. When the isthmus was compared with the infundibulum and ampulla, the down-regulated pathways were protein processing in the endoplasmic reticulum and the endocytosis. The Epstein–Barr virus infection pathway was up-regulated according to a comparison of the isthmus and infundibulum and was down-regulated based on a comparison of the isthmus and ampulla. Transcriptional misregulation in the Middle East pathway was up-regulated based on a comparison of the isthmus and ampulla and was down-regulated based on a comparison of the isthmus and infundibulum. ERBB2, JUP, CTNND1, and KRT7 were defined as the hub genes of the yak oviduct. The results of this study provide sufficient omics data for yak fertilization, which is also of great significance to altitude medicine.

Leukemia inhibitory factor (LIF) is a multipotent cytokine of the IL-6 family which plays a critical role in the maturation and development of oocytes. This study evaluated the influence of LIF on the maturation and development ability of yak oocytes, and the quality of subsequent blastocysts under in vitro culture settings. Different concentrations of LIF (0, 25, 50, and 100 ng/mL) were added during the in vitro culture of oocytes to detect the maturation rate of oocytes, levels of mitochondria, reactive oxygen species (ROS), actin, and apoptosis in oocytes, mRNA transcription levels of apoptosis and antioxidant-related genes in oocytes, and total cell number and apoptosis levels in subsequent blastocysts. The findings revealed that 50 ng/mL LIF could significantly increase the maturation rate (p < 0.01), levels of mitochondria (p < 0.01) and actin (p < 0.01), and mRNA transcription levels of anti-apoptotic and antioxidant-related genes in yak oocytes. Also, 50 ng/mL LIF could significantly lower the generation of ROS (p < 0.01) and apoptosis levels of oocytes (p < 0.01). In addition, blastocysts formed from 50 ng/mL LIF-treated oocytes showed significantly larger total cell numbers (p < 0.01) and lower apoptosis rates (p < 0.01) than the control group. In conclusion, the addition of LIF during the in vitro maturation of yak oocytes improved the quality and the competence of maturation and development in oocytes, as well as the quality of subsequent blastocysts. The result of this study provided some insights into the role and function of LIF in vitro yak oocytes maturation, as well as provided fundamental knowledge for assisted reproductive technologies in the yak.

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.

In this study, we detected the expression of mRNAs, lncRNAs, and miRNAs in primary cultured leydig cells (LCs) and sertoli cells (SCs) of yak by RNA sequencing technology. A total of 84 differently expression mRNAs (DEmRNAs) (LCs vs. SCs: 15 up and 69 down), 172 differently expression lncRNAs (DElncRNAs) (LCs vs. SCs: 36 up and 136 down), and 90 differently expression miRNAs (DEmiRNAs) (LCs vs. SCs: 72 up and 18 down) were obtained between the two types of cells. GO enrichment and KEGG analysis indicated that the differential expression genes (DEGs) were more enriched in the regulation of actin cytoskeleton, Rap1/MAPK signaling pathway, steroid biosynthesis, focal adhesion, and pathways associated with metabolism. Targeted regulation relationship pairs of 3β-HSD and MSTRG.54630.1, CNTLN and MSTRG.19058.1, BRCA2 and MSTRG.28299.4, CA2 and novel-miR-148, and ceRNA network of LAMC3-MSTRG.68870.1- bta-miR-7862/novel-miR-151/novel-miR-148 were constructed by Cytoscape software. In conclusion, the differences between LCs and SCs were mainly reflected in steroid hormone synthesis, cell proliferation and metabolism, and blood-testicular barrier (BTB) dynamic regulation, and 3β-HSD, CNTLN, BRCA2, CA2, and LAMC3 may be the key factors causing these differences, which may be regulated by ncRNAs. This study provides a basic direction for exploring the differential regulation of LCs and SCs by ncRNAs.

Fas-associated factor 1 (FAF1), a member of the Fas family, is involved in biological processes such as apoptosis, inflammation, cell proliferation and proteostasis. This study aimed to explore the biological role of FAF1 in testicular tissue at different ages (juveniles (1 and 2 years old), adults (3, 4, 6, and 7 years old) and old-aged animals (11 years old)) and ovaries during different reproductive cycle phases (follicular, luteal, and pregnancy phases). FAF1 mRNA, relative protein expression and protein expression localization were determined in testes and ovaries using real-time quantification, WB and immunohistochemistry (IHC), respectively. Real-time quantification of testis tissues showed that the relative expression of FAF1 mRNA in testis tissues at 3, 4 and 7 years of age was significantly higher than of those in other ages, and in ovarian tissues was significantly higher in luteal phase ovaries than those in follicular and pregnancy phase ovaries; follicular phase ovaries were the lowest. WB of testis tissues showed that the relative protein expression of FAF1 protein was significantly higher at 11 and 7 years of age; in ovarian tissue, the relative protein expression of FAF1 protein was significantly higher in follicular phase ovaries than in luteal and pregnancy phase ovaries, and lowest in luteal phase ovaries. The relative protein expression of FAF1 at 3, 4 and 7 years of age was the lowest. IHC showed that FAF1 was mainly expressed in spermatozoa, spermatocytes, spermatogonia and supporting cells; in ovarian tissue, FAF1 was expressed in ovarian germ epithelial cells, granulosa cells, cumulus cells and luteal cells. The IHC results showed that FAF1 mRNA and protein were significantly differentially expressed in testes of different ages and ovarian tissues of different reproductive cycle phases, revealing the significance of FAF1 in the regulation of male and female B. grunniens reproductive physiology. Furthermore, our results provide a basis for the further exploration of FAF1 in the reproductive physiology of B. grunniens.

Exosomes in the follicular fluid can carry and transfer regulatory molecules to recipient cells, thus influencing their biological functions. However, the specific effects of yak follicular fluid exosomes on 2-hydroxyestrodiol (2-OHE2) secretion remain unknown. Here, we investigated whether yak follicular fluid exosomes can increase 2-OHE2 secretion through the activation of autophagy in cumulus cells (YCCs). In vitro cultured YCCs were treated with yak follicular fluid exosomes for 6, 12, and 24 h. The effects of yak follicular fluid exosomes on autophagy and 2-OHE2 secretion were evaluated through real-time quantitative fluorescence PCR (RT-qPCR), Western blotting (WB), transfected with RFP-GFP-LC3, immunohistochemistry, and ELISA. To further investigate whether 2-OHE2 secretion was related to autophagy, YCCs were administered with yak follicular fluid exosomes, 3-methyladenine (3-MA), and rapamycin (RAPA). The results revealed that treatment with yak follicular fluid exosomes activated autophagy in YCCs and increased 2-OHE2 secretion. Conversely, the inhibition of autophagy with 3-MA blocked these effects, suggesting that autophagy has an important role in 2-OHE2 secretion in YCCs. Treatment of YCCs with rapamycin showed similar results with yak follicular fluid exosomes as there was an increase in 2-OHE2 secretion due to the activation of autophagy in the treated cumulus cells. Our results demonstrate that autophagy is enhanced by yak follicular fluid exosomes, and this is associated with an increase in 2-OHE2 secretion in YCCs.

Hsp70 and Hsp90 play an important role in testis development and spermatogenesis regulation, but the exact connection between Hsp70 and Hsp90 and metabolic stress in cattle is unclear. Here, we focused on the male cattle–yak and yak, investigated the expression and localization of Hsp70 and Hsp90 in their tissues, and explored the influence of these factors on development and metabolism. In our study, a total of 54 cattle (24 cattle–yaks and 30 yaks; aged 1 day to 10 years) were examined. The Hsp90 mRNA of the cattle–yak was first cloned and compared with that of the yak, and variation in the amino acid sequence was found, which led to differences in protein spatial structure. Using real-time quantitative PCR (RT-qPCR) and Western blot (WB) techniques, we investigated whether the expression of Hsp70 and Hsp90 mRNA and protein are different in the cattle–yak and yak. We found a disparity in Hsp70 and Hsp90 mRNA and protein expression in different non-reproductive organs and in testicular tissues at different stages of development, while high expression was observed in the testes of both juveniles and adults. Moreover, it was intriguing to observe that Hsp70 expression was significantly high in the yak, whereas Hsp90 was high in the cattle–yak (p < 0.01). We also examined the location of Hsp70 and Hsp90 in the testis by immunohistochemical (IHC) and immunofluorescence (IF) techniques, and the results showed that Hsp70 and Hsp90 were positive in the epithelial cells, spermatogenic cells, and mesenchymal cells. In summary, our study proved that Hsp70 and Hsp90 expressions were different in different tissues (kidney, heart, cerebellum, liver, lung, spleen, and testis), and Hsp90 expression was high in the testis of the cattle–yak, suggesting that dysplasia of the cattle–yak may correlate with an over-metabolism of Hsp90.

Abstract 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.

Due to its rich nutritional value, yak milk is an important food source in the alpine pastoral areas. However, yaks have a low milk yield. The Hippo pathway participates in cell proliferation and organ development. We aimed to determine the regulatory mechanism of this pathway in yak mammary cells. A greater understanding of how the expression of its essential genes influence the reproductive cycle could lead to improvements in lactation performance. The expression levels of the key genes MST1, LATS1, YAP1, and TEAD1 were detected by quantitative real-time PCR, Western blotting, and immunohistochemistry during the growth, lactation, and dry periods (GP, LP and DP, respectively). The MST1 and LATS1 mRNA and protein expression level was highest during GP and lowest during LP. The YAP1 and TEAD1 mRNA and protein expression level decreased from GP to LP and DP. MST1 and LATS1 were expressed in the cytoplasm whereas YAP1 and TEAD1 were expressed in the nucleus and cytoplasm, respectively. The differential expression of MST1, LATS1, YAP1, and TEAD1 expression in the yak mammary gland during different developmental stages strongly suggests that they play an important role in the regulation of developmental functions under different physiological conditions.

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 estrogen-signalling pathway is critical for normal follicular development; however, little is known about its importance during in vitro maturation (IVM) in large animals, particularly yaks (Bos grunniens). Through the present study, we aimed to determine the mechanisms underlying estrogen involvement in cumulus expansion and the subsequent development of cumulus-oocyte complexes (COCs). COCs were cultured in the maturation medium supplemented with different concentrations (10-6-10-3 mM) of 17β-estradiol (E2) or its receptor antagonist, fulvestrant, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot were performed to determine the expression of cumulus-expansion related factors and oocyte-secreted factors (OSFs). The cumulus expansion of COCs was observed using an inverted microscope, and COCs developmental ability were judged by the evaluation of cleavage and blastulation rates per inseminated oocytes by IVF, and the number of cells in the blastocyst. Cumulus expansion increased with 10-6-10-3 mM E2, but decreased with fulvestrant. HAS2, PTGS2, PTX3 and OSFs expression increased in the 10-6-10-3 mM E2 groups. Significantly higher cleavage and blastocyst rates were observed in the 10-4 mM E2 group than in the fulvestrant and 0 mM E2 groups. Moreover, in the 10-4 mM group, blastocysts at 7 days had higher cell counts than the other groups. In conclusion, the increase in cumulus expansion and subsequent oocyte development after the addition of E2 to IVM medium may have resulted from increased cumulus-expansion-related factor expression and OSF levels.

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, ≤2 years juvenile (y-c, n = 3; yak, n = 3) and ≥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.

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’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’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’s main reproductive organs during different stages implies that they play an important role in regulating the reproductive function under different physiological statuses.

The aim of this study is to investigate the expression and localization of HSP70/90 in different tissues and explore the regulation effects of HSP70/90 at lactation period of female yaks. HSP90 mRNA was cloned from the heart samples of female yaks, Quantitative real-time (qRT-PCR), Western blotting (WB), immunohistochemistry and immunofluorescence assays were utilized to analyze the expressions of HSP70/90 mRNA and protein in different tissues. Sequence analysis showed that HSP90 is a conserved molecular chaperone of female yaks. The qRT-PCR, WB results showed that the expressions of HSP70/90 mRNA and protein were significantly different in different tissues, and 3-fold higher expression during the lactation period than the non-lactation period of breast tissue (P < 0.01). Immunohistochemistry and immunofluorescence assays results showed that HSP70/90 were located in the cardiac muscle cells, cerebellar medulla, theca cells lining at the reproductive system, and the mammary epithelia of the breasts. In addition, the expression level of HSP70 was higher than those of HSP90 in all examined tissues. Therefore, our results strongly suggest that the expression and localization of HSP70/90 could provide significant evidence to further research in tissue specific expression, and lactation function of female yaks.

The study aimed to identify the changes of anatomic and microscopic structure and the expression and localization of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in the myocardium and coronary artery of the yak heart adapted to chronic hypoxia with aging. Thirty-two yaks (1 day, 6 months, 1 year, 2 years, and 5 year old) were included, and immunoelectronmicroscopy, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used. Right ventricular hypertrophy was not present in yaks with aging. There was no intima thickening phenomenon in the coronary artery. The ultrastructure of myofibrils, mitochondria, and collagen fibers and the diameter and quantity of collagen changed significantly with aging. The enzymatic activity of complexes I, II, and V increased with age. Immunogold labeling showed the localization of HIF-1α protein in the cytoplasm and nuclei of endothelial cells and cytoplasm of cardiac muscle cells, and VEGF protein in the nuclei and perinuclei areas of smooth muscle cells of coronary artery, and in the cytoplasm and nuclei of endothelial cells. ELISA results showed that HIF-1α secretion significantly increased in the myocardium and coronary artery from an age of 1 day to 2 years of yaks and decreased in old yaks. However, VEGF protein always increased with aging. The findings of this study suggest that 6 months is a key age of yak before which there are some adaptive changes to deal with low-oxygen environment, and there is a maturation of the yak heart from the age of 6 months to 2 years.