Objective To describe the phenotype of NK cells in Bama miniature pigs, and establish an efficient activation and culture method for porcine cytokine-induced killer ( CIK) cells in vitro. Methods The porcine peripheral blood mononuclear cells ( PBMCs) were isolated by Percoll gradient centrifugation, and the phenotype of NK cells was test-ed by detecting the CD2 + /CD8 + /CD3 - cell compartment. To establish an efficient activation and culture method for por-cine CIK cells, we optimized the culture conditions to improve the CIK activation efficiency. Results Using the optimized induction culture conditions, the ratio of CIK ( CD2 + /CD8 + /CD3 -) cells was up to 43. 63% at the fifth day, approxi-mately 5. 59 times increased compared with the initially separated PBMCs. Cell proliferation experiments showed that three obvious fluorescence peaks were observed on the fifth day. The results indicated that the induced CIK cells underwent three times cell division, in theory, about increased 8-fold compared with the initial separation of PBMCs. Furthermore, the qRT-PCR result of the surface markers of porcine NK cells also showed a similar variation tendency as the flow cytometry results. Conclusions Our findings demonstrate the successful establishment of an efficient activation and culture method for porcine CIK cells in vitro.

Objective To establish a breeding method ofMyodes rufocanus in the laboratory,collect their growth and reproduction data,and provide a basis for carrying out the experimental animalization.Methods Wild Myodes rufocanus caught in the Moranbong woodland were brought back to the laboratory.They were bred artificially in a large hard wall rodent negative pressure isolator.Their growth and reproduction data were recorded for evaluating the results of breeding.Results The Myodes rufocanus were successfully bred in the laboratory.The pregnancy rate was 54.55%.The average pregnancy length was 20.4 days(8 to 22 days).During one breeding period,they gave birth 2.9 times on average.The maximum number of births was 7 times,far more than the number tested under field conditions.The average litter size was 4.3±1.22.The highest litter number of a single nest was 8.The weaning rate of pups was 94.8%.The growth and development of pups were good.Conclusions The breeding method for Myodes rufocanus is established.The growth and reproduction data are tested too.The results of our study laid a foundation for the experimental animalization of Myodes rufocanus.

Objective Grey red-backed voles (Myodes rufocanus) are agile, fierce and hard to catch, thus, it is difficult to judge their gender by external appearance, especially for the juvenile voles. Therefore, it may cause difficulties to their allocation and later breeding in laboratories. The aim of this paper is to establish a rapid, simple and accurate method for gender identification of grey red-backed voles. Methods Fresh hair follicles were taken from 6 adult male voles, 3 adult females and 14 4-week-old juvenile voles, 5 male and 5 female 9-week-old Wistar rats, and 5 male and 3 female 6-week-old BALB/c mice. The genomic DNA was extracted using Chelex-100 resin and the zinc-finger Y/X gene (ZFY/ZFX) and the gene of sex-determining region of the Y (SRY) chromosome were amplified by PCR, and a double PCR amplification method was established. Results The ZFY/ZFX gene and SRY gene were simultaneously amplified from the male voles, while only the ZFY/ZFX gene was amplified from the females. The gender of all 23 voles, 10 Wistar rats and 8 BALB/c mice were correctly identified with this method, and the PCR results were consistent with the phenotypic and autopsy results. Conclusions Using fresh hair follicles as experimental materials for gender identification of grey redbacked voles can alleviate shock and damage to the animals. The established double PCR amplification method is accurate, simple, rapid, and deserves to be used for gender identification of grey red-backed voles.

Objective Based on the liquid chromatography-tandem mass spectormetry (LC-MS/MS), to study the effects of Pogostemon cablin on serum metabolism of Guizhou miniature pigs， and to explore its pharmacological mechanism.MethodsNine healthy Guizhou miniature pigs were divided into two groups, namely Pogostemon cablin drug group (n=5) and control group (n=4). The pigs in Pogostemon cablin drug group were orally fed with traditional Chinese medicine formula granules, each 0.5 g per day, for consecutive 8 days, while those in control group were given normal feeding without additional treatment. After the feeding experiment, serum samples were collected and analyzed using the LC-MS/MS technology. The metabolomics data was annotated and compared with KEGG, HMDB and Lipidmaps databases. Bioinformatics analysis methods including partial least squares discriminant analysis (PLS-DA), intergroup clustering, differential metabolite analysis and functional enrichment were used to screen differential metabolic biomarkers and their possible metabolic pathways.Results Forty-four differential metabolites (P<0.05) were screened out from the 443 metabolites, eight differential metabolites were significantly up-regulated (P<0.01), namely cinnamoylglycine, N-benzyl-N-isopropyl-N'-[4-(trifluoromethoxy) phenyl]urea, hypotaurine, D-glucose 6-phosphate, cis-2-decenoic acid, 11(Z),14(Z)-eicosadienoic acid, prostaglandin A2 and 10-hydroxydecanoic acid, and three differential metabolites were significantly down-regulated (P<0.01), namely lysophosphatidyl choline 22:5, lysophosphatidic acid 22:6 and lysophosphatidic acid 22:5. The differential metabolites were mainly enriched in the metabolic pathways of alanine, aspartate and glutamate metabolism (MapID: map00250) and taurine and hypotaurine metabolism (MapID: map00430).Conclusion Pogostemon cablin can significantly affect the metabolism of lysophosphatidic acids in porcine, and relieve the disorder of amino acids metabolism and regulate the occurrence of inflammation by affecting the metabolic pathways of alanine, aspartate and glutamate metabolism and taurine and hypotaurine metabolism.