Seasonal estrus is an important factor limiting the fertility of some animals such as sheep. Promoting estrus in the anestrus season is one of the major ways in improving the fecundity of seasonally breeding animals. The pineal-hypothalamus-pituitary-ovary (PHPO) axis plays a decisive role in regulating animal reproduction. However, the molecular mechanisms by which the PHPO axis regulates seasonal reproduction in animals are not well understood, especially in Tan sheep. To this end, we collected pineal, hypothalamus, pituitary and ovary tissues from Tan sheep during estrus and anestrus for RNA-Sequencing, and performed bioinformatics analysis on the entire regulatory axis of the pineal-hypothalamic-pituitary-ovary (PHPO). The results showed that 940, 1,638, 750, and 971 DEGs (differentially expressed genes, DEGs) were identified in pineal, hypothalamus, pituitary and ovary, respectively. GO analysis showed that DEGs from PHPO axis-related tissues were mainly enriched in “biological processes” such as transmembrane transport, peptide and amide biosynthesis and DNA synthesis. Meanwhile, KEGG enrichment analysis showed that the bile acid secretion pathway and the neuroactive ligand-receptor interaction pathway were significantly enriched. Additionally, four potential candidate genes related to seasonal reproduction (VEGFA, CDC20, ASPM, and PLCG2) were identified by gene expression profiling and protein-protein interaction (PPI) analysis. These findings will contribute to be better understanding of seasonal reproduction regulation in Tan sheep and will serve as a useful reference for molecular breeding of high fertility Tan sheep.

Neospora caninumis an important apicomplexan parasite causing neosporosis in cattle. The disease is recognized as one of the most important cause of reproductive problems and abortion in cattle worldwide. In this context, we developed an indirect enzyme-linked immunosorbent assays (ELISA) with chimeric protein rSRS2-SAG1-GRA7 to diagnose antibodies toNeospora-infection. This indirect ELISA was compared to indirect fluorescent antibody test (IFAT) and western blotting (WB), and the sensitivity and specificity results of ELISA were calculated to be 86.7 and 96.1%, respectively. The overall coincidence rate was 92.6% using IFAT and WB. Additionally, 329 aborting dairy cattle serum samples were tested using this ELISA to evaluate the prevalence ofN. caninumin Ningxia, China. The positive rate ofN. caninumin these farms was from 19.05 to 57.89%, and the mean rate was 41.64% (±11.01%), indicating that infection withN. caninummay be one of the important causes of cattle abortion in this region. This established rSRS2-SAG1-GRA7 indirect ELISA is capable for detecting the antibodies againstN. caninum, and it could be a useful screening tool for monitoring the epidemiology of neosporosis in cattle.

Theileria annulata schizont-infected host cells in culture in vitro show unlimited proliferation similar to tumor cells; thus far, T. annulata and T. parva are the only eukaryotes that have been found to transform mammalian cells (immortalized). The transformation of these cells is reversible; when the parasite is eliminated in transformed cells by buparvaquone (BW720c), the host cells show normal growth and apoptosis. TFG is a tropomyosin-receptor kinase fused gene that is conserved among many species and is an important proto-oncogene. In this study, the bovine TFG gene was amplified by PCR from the cDNA of T. annulata schizont-transformed cells, cloned into the pGEX-4T-1 vector and expressed in Escherichia coli BL21 (DE3). After purification, the fusion protein was injected into rabbits to produce polyclonal antibodies. Using T. annulata-transformed cells together with BW720c treatment to kill the parasite, we aimed to identify changes in TFG gene expression by real-time PCR and Western blotting. The results showed that the bovine TFG gene was ~582 bp in size; SDS-PAGE analysis showed that the fusion protein was expressed in BL21 (DE3) cells with a molecular mass of 48 kD, and Western blotting indicated that the polyclonal antibodies could react with bovine TFG proteins from T. annulata-transformed cells and showed high specificity. Compared with that in the control group, the transcription level of the host TFG gene decreased significantly in the BW720c test group, and the expression of host tumor-related TFG protein decreased sharply after 72 h of drug treatment, suggesting that the TFG protein expression in transformed cells was directly related to T. annulata. This finding laid a foundation for further study on the interaction between T. annulata and host cells.

Piroplasmosis and anaplasmosis are important zoonotic diseases of animal origin, which can be transmitted by ticks to infect animals. However, there is limited information on the infection of piroplasmosis and anaplasmosis in Tan sheep in Ningxia, China. In order to understand the prevalence of piroplasmosis and anaplasmosis in Tan sheep in Ningxia, 150 blood samples of Tan sheep from farms in five urban areas of Ningxia were detected by PCR, and some positive samples were sequenced to establish a phylogenetic tree. PCR revealed that the prevalence of Anaplasma spp. in Tan sheep in Ningxia was 28.0%. The overall prevalence of Piroplasmids was 33.3%, of which Theileria spp. and Babesia spp. were 20.7% and 12.7%, respectively. Among the samples of different ages, the highest detection rates of Piroplasmids and Anaplasma spp. were found in Tan sheep aged 20–30 months, and the detection rate of Theileria spp., Babesia spp., and Anaplasma spp. were 25.4%, 23.6%, and 36.3%, respectively. In this study, one Theileria species was identified as Theileria uilenbergi, two Babesia species were identified as Babesia molasi and Babesia ovis, and two Anaplasma species were identified as Anaplasma ovis and Anaplasma phagocytophilum, and the dominant species were A. ovis and T. uilenbergi. To the best of our knowledge, this is the first report detailing the infection rate and genotype of Piroplasmids and Anaplasma spp. in Tan sheep in Ningxia, China. The results of this study provide valuable data for understanding the epidemiology of tick‐borne disease in Tan sheep in Ningxia, China, and lay a theoretical foundation for the prevention and control of piroplasmosis and anaplasmosis in Tan sheep in Ningxia, northwest China.