Cattle-yak milk is an important raw material and an indispensable source of high-quality food for local farmers and herdsmen to produce ghee, milk residue, yogurt, and other dairy products. In this study, Lactobacillus strains were isolated from cattle-yak milk for potential probiotic candidates using a series of in vitro tests, including probiotic characterization and safety evaluation (antibiotic susceptibility and hemolytic ability). The results found that the Lactobacillus rhamnosus CY12 strain showed a high survival rate in bile salts, under acid conditions, and in the gastrointestinal juice environment, as well as showing high antimicrobial activity and adhesive potential. The safety evaluation showed that all strains were considered non-hemolytic. In addition, the whole-genome sequencing indicated that the strain CY12 spanned 2,506,167 bp, with an average length of 881 bp; the GC content in the gene region (%) was 47.35, contained 1347 protein-coding sequences, and accounted for 85.72% of the genome. The genome annotation showed that genes mainly focused on the immune system process, metabolic process, carbohydrate utilization, carbon metabolism, galactose metabolism, and biological adhesion, etc. This study revealed that the Lactobacillus rhamnosus CY12 strain might be an excellent potential probiotic in the development of feed additives for animals and has the ability to promote health.

Purpose: Endometritis is a female reproductive disease that affects the cattle industries development and microRNAs (miRNAs) play a pivotal role and critical regulators of the innate immune response in varieties of diseases. The present study intends to investigate the regulatory role of miR‐24-3p in the innate immune response involved in endometritis and evaluate its therapeutic potential. Methods: Whole mice uteri and bovine endometrial epithelial cells (BEECs) were separately stimulated with LPS. The BEECs were also transfected with miR-24-3p mimic and negative control; siTRAF6 and siNC; pcDNA3.1 empty and pcDNA3.1(+)TRAF6 separately with LPS stimulation. The expression levels of miR‐24-3p and TRAF6 were measured via quantitative real‐time polymerase chain reaction (qRT-PCR) and Western blot, respectively. LPS‐induced inflammatory response assessed by inflammatory cytokines secretion and expression via ELISA and qRT-PCR. Bioinformatics analysis and luciferase reporter assay validated the interaction between miR‐24-3p and TRAF6. The activation of the NF‐ĸB/MAPK pathway and p65 phosphorylation was investigated by Western blot and immunofluorescence assay, respectively. Results: The expression of miR‐24-3p was decreased, and TRAF6 was elevated with an increased level of pro-inflammatory cytokines in LPS‐treated BEECs and mice uterus. The overexpression of miR‐24-3p suppressed LPS‐induced secretion of inflammatory cytokines (IL‐1β, IL‐6, IL-8 and TNF-α) and deactivation of NF‐ĸB/MAPK pathways. The downregulation of TRAF6 inhibited LPS‐induced inflammatory response in BEECs. TRAF6 is validated as a target of miR‐24-3p, and miR‐24-3p reversed the overexpression of cloned TRAF6 on inflammation response in BEECs. Conclusion: Our findings demonstrate that the overexpression of miR‐24-3p attenuates endometrial inflammation and the expression of pro-inflammatory mediators via suppressing TRAF6. Therefore, modulating the pathogenesis of endometritis and possibly, a therapeutic potential against endometritis.

The complex etiology, higher morbidity and mortality, poor prognosis, and expensive cost of calf diarrhea have made it a catastrophic disease in the dairy industry. This study aims to assess the biomarkers in calves with diarrhea and to predict the biomarkers related to the pathway. As subjects, nine calves with diarrhea and nine healthy calves were enrolled, according to strict enrollment criteria. The serum metabolites were detected by a liquid chromatographic tandem mass spectrometry (LC-MS/MS), and then analyzed by online multivariate statistical analysis software to further screen the biomarkers. In addition, the biomarkers involved in the metabolic pathways of calves with diarrhea and healthy calves were analyzed. In the serum of calves with diarrhea, nine biomarkers were found to which several biomarkers exhibited a certain relation. Moreover, these biomarkers were involved in important metabolic pathways, including protein digestion and absorption, ABC transporters, aminoacyl-tRNA biosynthesis, mineral absorption, and fatty acid biosynthesis. All these findings suggested that the imbalance of these markers was closely related to the occurrence and development of calf diarrhea. The targeted regulation of metabolic pathways involved in these biomarkers may facilitate the diagnosis, treatment, and discussion of the mechanism of calf diarrhea.

Neutrophils are involved in the development of endometritis, but it remains unknown how neutrophils induce inflammation and tissue damage. Neutrophil extracellular traps (NETs) clear invading pathogens during infection but induce pyroptosis, leading to inflammation and tissue damage. Thus, our objective was to investigate whether NETs participate in bovine endometrial epithelial cell (BEEC) pyroptosis during endometritis. To confirm this, NETs and caspase-1/4; apoptosis-associated speck-like protein containing a caspase-recruitment domain(ASC); nod-like receptor protein-3 (NLRP3); and gasdermin D N-terminal (GSDMD-N), TNF-a, IL-1β, IL-6, and IL-18 in endometrial tissue were detected. Pathological section and vaginal discharge smears were performed to visually determine endometritis in the uterus. BEECs were stimulated with NETs to induce pyroptosis, which was treated with DNase I against pyroptosis. Caspase-1/4, ASC, NLRP3, GSDMD-N, TNF-a, IL-1β, IL-6, and IL-18 in BEECs were analyzed in endometrial tissue. The results showed that NET formation, as well as pyroptosis-related proteins and proinflammatory, cytokines were elevated in the endometrial tissue of cows with endometritis. Pathological sections and vaginal discharge smears showed increased neutrophils and plasma cells in the uterus, as well as tissue congestion. In BEECs, NETs increased the level of pyroptosis-related proteins and proinflammatory cytokines and were diminished by DNase I. In summary NETs participate BEEC pyroptosis during endometritis in dairy cows.

Objective: Streptococcus dysgalactiae is a major pathogen in bovine mastitis. The purpose of this study was to survey the prevalence, antimicrobial resistance, as well as the spread of resistance and virulence-associated gene of S. dysgalactiae. Methods: A total of 60 S. dysgalactiae strains were obtained from 830 milk samples from Holstein cows with clinical mastitis. Antimicrobial resistance was examined by the disk diffusion method. Antimicrobial resistance and virulence genes were investigated by PCR, agarose gel electrophoresis and 16S rRNA gene sequencing. Results: All isolates were resistant to tetracycline and showed a high level of resistance to aminoglycoside antibiotics, where 81.67% of the strains were multi-resistant to these ten sorts of antibiotics. In addition, the most prevalent resistance gene in S. dysgalactiae was aphA-1 (98.33%), followed by blaTEM (96.67%), ermB (83.3%), aadA1/aadA2 (78.33%) and tetL (73.33%). Totally, seven virulence genes with 25 combination patterns were detected in these isolates, and each isolates harbored at least one virulence gene. 21.67% of the isolates carried three or more virulence genes, while one strain with seven virulence-related genes and belonged to cfb+lmb+eno+napr+bca+scpB+cyl. Conclusion: These findings indicate that S. dysgalactiae isolated from clinical bovine mastitis cases in Northwest China show a variety of molecular ecology and are highly resistant to antibiotics commonly used in dairy farms. This research will help investigators better understand the pathophysiology S. dysgalactiae in bovine mastitis and choose the appropriate antibiotics to treat mastitis.