Chunxia Liu

This study focuses on optimizing the liquid preservation protocol for Mongolian sheep semen at 17 °C. We systematically compared the protective effects of eight extender formulations on sperm quality and analyzed dynamic metabolic changes under optimal storage conditions via LC-MS/MS metabolomics, aiming to provide experimental evidence for improving semen preservation techniques and to establish a foundation for the selection of preservation media in future germplasm conservation efforts. By comparing eight diluents, a semen preservation formulation for Mongolian sheep was optimized. The eighth diluent, including fructose, lactose, and Tris, effectively sustained sperm motility during liquid storage at 17 °C. To clarify the underlying metabolic mechanism, LC-MS/MS metabolomics identified 2669 metabolites in spermatozoa at storage times of 0, 48, 96, and 144 h. Based on the metabolomics analysis of differential metabolites, three antioxidants including vitamin E (acting as the basic antioxidant protection), lycopene (for targeted inhibition of lipid peroxidation) and resveratrol (suggested to have endogenous protective effects) were used for exogenous addition experiments. Subsequently, the optimal concentrations for addition were screened: resveratrol (25 μM), lycopene (5 μM), and vitamin E (1.5 μM). Verification experiments revealed that each of the three antioxidants could independently sustain sperm viability above 50% for up to 7 days. Moreover, the combined supplementation exhibited a synergistic effect, increasing sperm viability to 55.05%. This study not only elucidates the underlying mechanism of sperm preservation at the metabolic level, but also provides a validated formulation basis and theoretical support for the development of efficient sheep semen preservatives.

Haemonchus contortus is widely distributed in the world, which is seriously harmful to small ruminants and has serious resistance to common anthelmintics. Over time, the prevalence and intensity of host infection with this parasite have gradually increased, so it is necessary to study the phylogenetic and genetic changes of single anthelmintic resistant strains. This study is the first to analyze the genetic diversity and population structure about IVM-resistant H.contortus (L3,egg, adult, n = 30 ) in Inner Mongolia, China. The H.contortus were identified to IVM resistance and cultured by fecal egg count reduction test, larval development inhibition test and larval locomotor behaviour test. The complete sequence of ITS2 ( second internal transcribed spacer ) and partial sequence of nad4 ( nicotinamide dehydrogenase subunit 4 gene ) in L3, adult and egg were amplified. At the same time, compared with the ITS2 and nad4 partial sequences of H.contortus reported in NCBI database to carry out phylogenetic analysis and haplotype determination, so as to further study the variation and population genetic diversity of IVM-RR samples. The samples were identified as H.contortus resistant to IVM. Phylogenetic analyses of ITS2 of IVM-RR and other H.contortus showed that they were from the same branch, genetic relationship was close, the intraspecific was relatively conserved, and no significant correlation with AR. There was no significant difference between different stages of development of the samples. In addition, from the 30 nad4 gene sequences of IVM-RR samples obtained 13 haplotypes, and the 169th base site in 70% of the nad4 gene was mutated. The nucleotide diversity was 0.00623 and haplotype diversity was 0.926. IVM-RR compared with five countries' H.contortus nad4, found that the genetic differentiation level of the IVM-RR population was relatively low with the China and Pakistan populations, and the Fixation index (Fst) were 0.10957 and 0.17262, respectively. The genetic differentiation levels with Bangladesh, Greece and France populations were higher, and the Fst values were 0.46978,0.61825 and 0.98101. Perhaps the population structure differentiation were correlated with regional localization and anthelmintic frequency treatment. In this report, we analyzed the genetic diversity and population differentiation of IVM resistant H.contortus at different developmental stages, and obtained resistant samples' haplotypes, hoping to provide a new direction for the study of AR and vaccine research.

Brucellosis is one of the most serious zoonotic bacterial diseases in the world. The disease has caused serious harm to people and livestock, hindered the healthy development of the breeding industry, and led to serious economic losses.At present, the prevention and control of this kind of disease is still based on vaccine immunization. However, after the widely used vaccine is inoculated to livestock, there is no widely used differential diagnosis method to distinguish vaccine immune antibodies from natural infection antibodies. Quarantine and purification work is difficult to carry out. In addition, there are few studies using real-time PCR(qPCR) methods in the differential diagnosis of natural virulent strains and vaccine strains of brucellosis.The purpose of this study is to establish a rapid, sensitive and accurate differential diagnosis method for Brucella S2 vaccine strain, and to solve the problem of lack of identification of Brucella S2 vaccine strain and natural virulent strain in clinical detection.It avoids the killing of some livestock due to the positive antibody of the Brucella S2 vaccine strain, and can also identify sick animals from immune herds, reducing the economic losses of farms, and providing certain technical support for the quarantine and purification of epidemic diseases. In this study, combined with TaqMan probe-based qPCR technology, specific primers and probes were designed according to the specific deletion genes of the Brucella S2 vaccine strain,which could be used as marker genes.The qPCR and duplex qPCR detection methods of Brucella were successfully established.The method has good specificity, sensitivity and repeatability, the lowest limit of detection can reach 1 × 101 copies/μL, the sensitivity is about 100 times higher than that of conventional PCR, and there is no cross-reaction with Escherichia coli,Salmonella,streptococcus and other common strains.The coefficient of variation between groups was less than 0.6%, and the coefficient of variation within groups was less than 0.55%.Subsequently, this method was used to monitor the antibody levels in goat inoculated with different doses of Brucella S2 vaccine strain, and the method could also detect the corresponding nucleic acid signals in goat milk samples, and the clinical samples were detected. In summary, this method has good specificity, sensitivity and repeatability, and can be used for the differential diagnosis of clinical brucellosis. This study successfully established a duplex qPCR detection method for the differential diagnosis of the Brucella S2 vaccine strain. From the establishment of the method to the clinical application of the method, it shows that the method can be used for the differential diagnosis of clinical brucellosis.

Haemonchus contortus has caused significant economic losses in many regions. The emergence of drug resistance has created new difficulties for the prevention and control of parasitic diseases in cattle and sheep. The mechanism of drug resistance to ivermectin in H. contortus is not clear; therefore, it is of great significance to study it. Non-coding RNAs, especially microRNAs (miRNAs), play important roles in drug resistance in some human tumors and insects. However, few studies have investigated whether miRNAs are involved in the regulation of drug resistance to ivermectin. In the early stages of this study, four miRNAs that may be involved in the regulation of ivermectin resistance in H. contortus were identified by high-throughput sequencing analysis of ivermectin-sensitive and -resistant strains of H. contortus. To verify whether these miRNAs are indeed related to ivermectin resistance in H. contortus, RNA interference (RNAi) experiments were performed on these miRNAs. The expression of candidate drug-resistance-related miRNA target genes before and after RNAi was detected by quantitative polymerase chain reaction. The resistance of the third-stage larvae (L3s) of H. contortus before and after RNAi was detected using 0.5% dimethyl sulfoxide and 100 µg/mL ivermectin treatment. The locomotion behavior of the L3s of H. contortus before and after RNAi was detected by counting the frequency of head thrashes and body bending. Finally, we confirmed that miR-10,025-y, miR-5960-z, miR-27-y, and miR-276-y were involved in and regulate ivermectin resistance in H. contortus.

BackgroundDifferent areas of sheep infected with different types of parasites, all will cause serious harm to the local sheep, and the widespread use and repeated use of anthelmintics have produced different degrees of anthelmintic resistance (AR) in various regions. We re-investigated the infection of common parasites and AR of Gastrointestinal Nematodes (GINs) in sheep in Horqin Right Wing Front Banner, and first investigated the common parasite types and AR of GINs in sheep at other four areas in Hinggan league (city), China.ResultsA total of 1770 fecal samples were collected from 1 prefecture-level city and 4 counties in Hinggan league, in which the infection rate of Coccidia ranged from 83.3% to 96.06%, that of Ascaris ovis ranged from 10.17% to 15.19%, that of Moniezia benedeni ranged from 0.6% to 1%, that of Moniezia expansa ranged from 0.33% to 8.15%. The infection rate of GINs was 100%, and Haemonchus contortus was still the dominant species. The AR results showed that only the closantel in Horqin Right Wing Middle Banner was low resiatant, and the other three regions had been resistant. Levamisole also occurred AR in the other four regions, the widely used ivermectin and albendazole had produced serious AR in five areas. The research shows that GINs are becoming more and more resistant to various anthelmintics, which has made the problem worse.ConclusionsUnderstanding the dynamic changes of parasite infections and the development trend of AR in sheep in the region in this paper, the development trend seems to be more serious than imagined. Therefore only by deeply understanding the parasitic infections of sheep in this land can more reasonable medication guidance be carried out. It is expected to provide new ideas formore innovative, scientific and sustainable methods of preventing and controlling parasites.

In order to find out an effective method for the in vivo diagnosis of parabronemosis and on the basis of results of histological analysis obtained in the laboratory, the serine threonine protein kinase (STPK) gene, an immune-related secretory gene of P. skrjabini, was screened. A total of 140 Bactrian camel sera samples from different regions of Inner Mongolia were screened. The results of bioinformatics analysis showed that the nucleic acid sequence of STPK gene was 579 bp, contained a complete open reading frame encoding 199 amino acids, had no signal peptide, was atypical secretory protein and had 8 antigenic epitopes. The total RNA of P. skrjabini was extracted and the STPK gene was obtained by RT-PCR amplification. The recombinant expression plasmid was constructed and expressed in Escherichia coli BL21 (DE3). A recombinant protein, rSTPK, 28 kDa in size, that was primarily expressed in inclusion bodies, was obtained. An indirect enzyme-linked immunosorbent assay (iELISA) using rSTPK as an antigen was established using recombinant protein. We observed a positive detection rate of 85.7% (120/140), indicating that rSTPK-iELISA can be used as a serological method to diagnose parabronemiasis in camels.