Comparison of Intestinal Microbiota of Blue Fox before and after Weaning.

Intestinal flora plays an important role in maintaining the internal stability and health of the intestine. Currently, intestinal microbes are considered an important "organ" but are mostly ignored by people. This study evaluated the flora structure of each intestinal segment of blue foxes pre-weaning and explored the differences between the fecal flora and intestinal flora structure of each segment after weaning. Samples of intestinal contents from three blue foxes at 45 days of age (before weaning) and intestinal contents and feces samples from at 80 days (after weaning) were collected for 16s rRNA flora analysis. The species and distribution characteristics of microorganisms in different intestinal segments of blue foxes before and after weaning were different. Except for the rectum, the dominant flora of each intestinal segment of blue fox changed significantly after experiencing weaning, and the fecal flora structure of young fox at the weaning stage did not represent the whole intestinal flora structure but was highly similar to that of the colon and rectum. To sum up, the intestinal flora of blue foxes changed systematically before and after weaning. When performing non-invasive experiments, the microflora structure of the colon and rectum of blue foxes can be predicted by collecting fecal samples.

Requirements and Metabolism for Calcium, Phosphorus and Vitamin D3 in the Growing-Furring Blue Foxes.

A 3 × 3 factorial experiment was conducted to investigate the influence of dietary calcium, phosphorus, and vitamin D3 (VD3) supplement levels on the growth performance, nutrient digestibility, and serum biochemical indices of growing-furring blue foxes. One hundred and thirty-five 120-day-old male blue foxes were randomly allocated into nine groups. The nine treatment diets were supplemented with 0%, 0.4%, or 0.8% Ca, and 1000, 2000, or 4000 IU·kg−1 VD3. The base diet contained 0.8% Ca and 327 IU·kg−1 VD3. The dietary calcium level had a significant effect on the average daily gain (ADG) of blue foxes at 121 to 135 days of age and 136 to 150 days (p < 0.05). The ADG of blue foxes at 121 to 135 days of age was significantly decreased by VD3 level (p < 0.05). The Ca dosage decreased the nutrient digestibility (p < 0.05). The Ca dosage increased the fecal Ca and P and decreased the P digestibility (p < 0.05). Interactions were found between the Ca and VD3 levels, which affected the digestibility of Ca and P (p < 0.05). In conclusion, this research determined the suitable doses of Ca and VD3 for growing-furring blue foxes.

Testicular Transcriptome of Males and Pseudo-Males Provides Important New Insight into Sex Reversal of Rana dybowskii.

Rana dybowskii (R. dybowskii) is an ecological species found in China, Japan, Korea, and Russia. Like most amphibians, R. dybowskii lacks heterotypic sex chromosomes, limiting the in-depth study of sex determination and sex reversal mechanisms. Previous studies have shown that certain environmental factors can modify R. dybowskii genotypic females into phenotypic males, but the mechanism is still unknown. Considering the difficulties in identifying and collecting sex reversal gonads at different stages of differentiation under natural conditions, testes from sexually mature wild adult R. dybowskii were taken in this study, and the genotypic sex of individuals and sex reversal were identified by two male-linked genetic markers reported in our most recent findings. Transcriptome sequencing was performed on testicular tissue from males and pseudo-males, as well as female ovary tissue. The results show that the gene expression patterns of pseudo-males' testes were similar to those of the males but highly differed from females' ovaries. One hundred and seventeen differentially expressed genes between testes of pseudo-males and males were found, and the up-regulation of doublesex and mab-3 related transcription factor 1 (Dmrt1) in testes of pseudo-males may play a key role in R. dybowskii sex reversal.

Progressive crushing 40Ar/39Ar dating of a gold-bearing quartz vein from the Liaotun Carlin-type gold deposit, Guangxi, southern China.

Carlin-type gold deposits are among the largest hydrothermal gold deposits in the world. However, direct dating the metallogenic age of these deposits is difficult, because not all deposits provide material suitable for conventional radiometric methods. Syn-mineralization stage quartz veins from these deposits usually contain abundant fluid inclusions, which allow fluid inclusion 40Ar/39Ar dating. In this study, progressive crushing 40Ar/39Ar dating has been performed on a gold-bearing quartz vein from the Liaotun Carlin-type gold deposit in northwestern Guangxi, China. Argon isotopes liberated from the later steps yielded an isochron age of 200.7 ± 2.1 Ma. We infer that Ar-bearing gas was extracted from the primary fluid inclusions, and that the age of ca. 200.7 Ma reflects the timing of gold mineralization. The initial 40Ar/36Ar ratio corresponding to the isochron is 298.0 ± 4.3, which is statistically indistinguishable from the value for air, indicating that the ore-forming fluids probably mainly derived from gravitational pressure flow in the basin of air-saturated water. Our preliminary study shows the feasibility and great potential of 40Ar/39Ar dating of gases from fluid inclusions by progressive crushing of quartz veins to date the mineralization age and decipher the fluid origins of Carlin-type gold deposits.

Effect of Dietary Supplementation with Calcium, Phosphorus and Vitamin D3 on Growth Performance, Nutrient Digestibility, and Serum Biochemical Parameters of Growing Blue Foxes.

Based on the randomized design, a 3 × 3 factorial experiment was designed to examine the effects of dietary calcium (Ca), phosphorus (P), and vitamin D3 (VD3) supplemental levels with a fixed 1.5/1 ratio of Ca to P on the growth performance, nutrient digestibility, and serum biochemical indices blue foxes' growth. In total, 135 male blue foxes with the age of 60 days were randomly divided into 9 groups each with 15 blue foxes. The blue foxes belonging to the nine treatment groups were fed Ca supplementation (0%, 0.4%, or 0.8%) and VD3 supplementation (1000, 2000, or 4000 IU/kg DM). The base diet contained 0.8% Ca and 327 IU/kg VD3. The dosage of VD3 in blue foxes showed a significant impact on their growth performance (p < 0.05). The Ca dosage had a linear effect on the digestibility of the CP and carbohydrates (CHO) (p < 0.05). In conclusion, the results indicated that the Ca and VD3 doses showed promising effects on growth performance and nutrient digestibility in growing blue foxes and could reduce fecal N and P via improvement in protein and P utilization.

Effects of Fly Maggot Protein Replacement of Fish Meal on Growth Performance, Immune Level, Antioxidant Level, and Fecal Flora of Blue Foxes at Weaning Stage.

Dietary protein is a key nutritional parameter and warrants special attention in animal husbandry. This study aimed to evaluate the effect of replacing fish meal (F) with fly maggot protein (M) on the growth performance, antioxidant levels, immune indexes, and fecal microflora in weaned blue foxes (Alopex lagopus). Twenty weaned blue foxes were randomly assigned to the control (F diet; 6% of F) or experimental (M diet; F substituted by M) group (10 blue foxes per group). The duration of the trial was 28 days. The results showed that there was no significant difference in average daily gain between group M and group F during the experiment (p = 0.473). Moreover, the diarrhea index was similar between group M and group F during the entire experimental period (p = 0.112). At the end of the experiment, the levels of IL-6 and IgG in group M at 28 d were significantly higher than that in group F (p = 0.004, p = 0.025, respectively), but not IL-1ß, IL-2, SIgA, IgM, and TNF-α. The levels of SOD in group M at 28 d were significantly higher than those in group F (p = 0.001), and no difference of MDA and T-AOC was found between group F and M (p = 0.073, p = 0.196, respectively). In both groups, the diversity of fecal microbes first increased and then decreased with the progress of the experimental period. Initially, there were differences in the composition of microbial communities between the two groups. However, this difference was attenuated at later stages of the experimental period. In conclusion, fly maggot protein can replace fish meal as a source of animal protein in feed material for blue foxes during the weaning period.

Clinical and molecular characteristics of myotonia congenita in China: Case series and a literature review.

Myotonia congenita (MC) is a rare genetic disease caused by mutations in the skeletal muscle chloride channel gene (CLCN1), encoding the voltage-gated chloride channel ClC-1 in skeletal muscle. Our study reported the clinical and molecular characteristics of six patients with MC and systematically review the literature on Chinese people. We retrospectively analyzed demographics, clinical features, family history, creatine kinase (CK), electromyography (EMG), treatment, and genotype data of our patients and reviewed the clinical data and CLCN1 mutations in literature. The median ages at examination and onset were 26.5 years (range 11-50 years) and 6.5 years (range 1.5-11 years), respectively, in our patients, and 21 years (range 3.5-65 years, n = 45) and 9 years (range 0.5-26 years, n = 50), respectively, in literature. Similar to previous reports, myotonia involved limb, lids, masticatory, and trunk muscles to varying degrees. Warm-up phenomenon (5/6), percussion myotonia (3/5), and grip myotonia (6/6) were common. Menstruation triggered myotonia in females, not observed in Chinese patients before. The proportion of abnormal CK levels (4/5) was higher than data from literature. Electromyography performed in six patients revealed myotonic changes (100%). Five novel CLCN1 mutations, including a splicing mutation (c.853 + 4A>G), a deletion mutation (c.2010_2014del), and three missense mutations (c.2527C>T, c.1727C>T, c.2017 G > C), were identified. The c.892 G > A (p.A298T) mutation was the most frequent mutation in the Chinese population. Our study expanded the clinical and genetic spectrum of patients with MC in the China. The MC phenotype in Chinese people is not different from that found in the West, while the genotype is different.

Male heterogametic sex determination in Rana dybowskii based on sex-linked molecular markers.

Identifying the mechanism for sex determination in amphibians is challenging. Very little is known about sex determination mechanisms of Rana dybowskii, a species of importance to evolutionary and conservation biology. We screened for sex-linked molecular markers in R. dybowskii in China using target region amplification polymorphism with 2 fixed primers against the sequences of Dmrt1. We found 2 male-linked molecular markers in R. dybowskii, which were 222 bp and 261 bp long. The detection rates of 222 bp marker in males form Xinglong, Huadian, and Dandong were 93.79%, 69.64%, and 13.64%, respectively, while the rate in females from Huadian was 27.50%. Besides, the detection rates of 261 bp marker in the above 3 regions were only observed in males at the rate of 93.79%, 87.50%, and 32.73%, respectively. The inheritance patterns of sex-linked molecular markers showed that the 2 sex-linked molecular markers were heterozygous. Compared to the XY-male parent, progeny from XX-pseudo-male parent possessed lower sex reversal ratio at the same rearing temperature, and the proportion of female froglets from an XX-pseudo-male parent was more than 95% at low rearing temperature (15°C). Our findings suggest that R. dybowskii displays male heterogamety, and the 2 sex-linked molecular markers may have a guiding significance for the protection and utilization of R. dybowskii.

Bone marrow stromal cells reverse the microglia type from pro-inflammatory tumour necrosis factor a microglia to anti-inflammatory CD206 microglia of middle cerebral artery occlusion rats through triggering secretion of CX3CL1.

The middle cerebral artery occlusion (MCAO) model has been extensively applied to study ischaemic stroke. This study attempted to clarify effect of bone marrow stromal cells (BMSCs) on infarct injury of MCAO rats. BMSCs were isolated and identified by staining CD29/CD44 and CD31/CD45. CX3CL1 silencing vector (pLVX-shRNA-CX3CL1) was generated and infected to BMSCs. pLVX-shRNA-CX3CL1 infected BMSCs were transplanted into brain tissue of MCAO rats. Real-time PCR was used to determine CX3CL1 expression. Infarct areas were stained with TTC to evaluate infarct size. Double-staining immunofluorescence was conducted to determine anti-inflammatory type CD206 and pro-inflammatory type tumour necrosis factor a (TNF-a) microglia. Isolated BMSCs were positively presented for CD29/CD44, and negatively for CD31/CD45. CX3CL1 was significantly lower in the BMSC + pLVX-shRNA2-CX3-CL1 group compared to the BMSCs + pLVX group (p < 0.05). According to TTC and neurological scores, MCAO rats were successfully generated. BMSCs transplantation significantly increased CD206 microglia and decreased TNF-a microglia. However, shRNA-CX3CL1-infected BMSCs remarkably reduced CD206 microglia and enhanced TNF-a microglia compared to the MCAO + BMSCs group. In conclusion, BMSCs reverse microglia from pro-inflammatory type TNF-a microglia to anti-inflammatory type CD206 microglia in the infarct region of MCAO rats (3rd to 7th days post BMSC transplantation), through triggering of CX3CL1 secretion. Therefore, the potential effects of CX3CL1 secreted by BMSCs would provide an insight for stem cell-dependent therapeutic strategies in treating ischaemic stroke-associated disorders.

Evaluation of Reference Genes for Quantitative PCR in Four Tissues from Rabbits with Hypercholesterolaemia

Abstract Rabbit with hypercholesterolaemia is an important model for studying cholesterol metabolism disease. This study aimed to evaluate the expression stability of nine reference genes for quantitative PCR (qPCR) analysis in adrenal gland, liver, spleen, and kidney tissue from rabbits with hypercholesterolaemia. In total, 30 male Harbin Large White (HLW) rabbits were fed a normal feed (n = 15) or a high cholesterol feed (n = 15) for 8 weeks to induce hypercholesterolaemia. Nine reference genes were verified by qPCR using cDNA extracted from rabbit tissue samples. For qPCR analysis, reference genes were evaluated using the RefFinder and GeNorm algorithms. Overall, seven rabbits with hypercholesterolaemia were identified based on body weight and total cholesterol measurements. Combining the results of the RefFinder and GeNorm algorithms, the most stable reference genes were hypoxanthine phosphoribosyltransferase 1 (Hprt1) and eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) in the adrenal gland, β-2-microglobulin (B2m) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) in the liver, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and Gapdh in the spleen, and peptidylprolyl isomerase (Ppia), β-actin (Actb), succinate dehydrogenase complex subunit A flavoprotein (Sdha), and B2m in the kidney. Taken together, our results confirmed that Hprt1 and Eef1a1, B2m and Gapdh, Ywhaz and Gapdh, and Ppia, Actb, Sdha, and B2m were the best reference genes for qPCR analyses in adrenal gland, liver, spleen, and kidney tissue, respectively, of rabbits with hypercholesterolaemia.

The 40Ar/39Ar dating of quartz: new insights into the metallogenic chronology of the Jinchang gold deposit and its geological significance.

The Jinchang gold deposit has been extensively studied, but precise dates for its formation are debated. Native gold mainly occurs as inclusions within pyrite and quartz. In this study, we analysed quartz crystals coeval with gold precipitation from two different types of mineralization using the ArgusVI multi-collector noble gas mass spectrometer by the stepwise crushing technique to resolve the timing and genesis of gold mineralization. 40Ar/39Ar dating of quartz samples (J12Q) from breccia ore yields a plateau age of 109.87 ± 0.86 Ma, and an inverse isochron age of 109.87 ± 0.88 Ma. Quartz samples (J18Q) from vein ore yields a slightly younger plateau age of 107.76 ± 0.85 Ma, with an inverse isochron age of 107.76 ± 0.92 Ma. These dates place the ore-forming age of the Jinchang gold deposit at 107~110 Ma, much younger than previously published radiometric ages, suggesting the gold mineralization is spatio-temporally associated with the granite porphyry. The formation of the Jinchang gold deposit is consistent with the regional late Mesozoic porphyry-epithermal gold mineralization event in the Yanbian-Dongning area. Finally, our study shows that 40Ar/39Ar of quartz can be used as a powerful tool to date the formation ages of hydrothermal ore deposits.

Five new species of the spider genus Pholcus Walckenaer, 1805 (Araneae: Pholcidae) from China and Thailand.

Five new species from four species-groups of the genus Pholcus Walckenaer, 1805 are described from China and Thailand: P. sakaew Yao Li sp. nov. (Thailand) from the P. bidentatus species-group; P. umphang Yao Li sp. nov. (Thailand) from the P. halabala species-group; P. ningan Yao Li sp. nov. (China) from the P. phungiformes species-group; and P. yongshun Yao Li sp. nov. (China) and P. yuxi Yao Li sp. nov. (China), both from the P. yichengicus species-group.

The complete mitochondrial genome sequence of Lindian Chicken (Gallus gallus) in China.

In this study, the first complete mitochondrial genome of Lindian Chicken (Gallus gallus) was sequenced in order to develop the mitogenome data for genus gallus. The complete mitogenome sequence is 16,785 bp in length, containing 37 genes (13 protein-coding genes, 2 ribosomal RNA, 22 transfer RNA genes, and one control region). The new sequenced complete mitogenome of Lindian Chicken will provide useful information for application in conservation genetics and evolution for this Near Threatened Chicken genomes.

[Application of A(2)DS(2) score for predicting post-stroke pneumonia in elderly patients].

OBJECTIVE: To investigate the risk factors for post-stroke pneumonia and assess the value of A(2)DS(2) score in predicting post-stroke pneumonia in elderly stroke patients. METHODS: The clinical data were retrospectively collected from elderly stroke patients from January, 2007 to December, 2012. A(2)DS(2) score was then assigned using the clinical information from the medical record. The ability of the score to discriminate between patients with post-stroke pneumonia and those without was quantified using ROC analysis. The calibration of the score was analyzed using Hosmer-Lemeshow goodness-of-fit test. RESULTS: A total of 131 elderly male stroke patients were enrolled in this study, among whom the incidence of post-stroke pneumonia was 29.01%. The independent risk factors for post-stroke pneumonia identified included moderate (P=0.0081, OR: 5.6089; 95%CI: 1.5663-20.0854) and severe (P=0.0048, OR: 44.4827; 95%CI: 3.1847-621.3126) neurological impairment, dysphagia (P=0.0005, OR: 7.5265; 95%CI: 2.4282-23.3292), and atrial fibrillation (P=0.0226, OR: 4.1778; 95%CI: 1.2221-14.2825). The incidence of post-stroke pneumonia ranged from 2.2% in patients with a A(2)DS(2) score less than 3 to 75% in those with a score higher than 8. The C-statistic of A(2)DS(2) score for predicting post-stroke pneumonia was 0.86 (95%CI: 0.784-0.911) by the ROC analysis. The A(2)DS(2) score was well calibrated to predict post-stroke pneumonia in elderly patients by Hosmer-Lemeshow test (7.083, P=0.528). CONCLUSION: The A(2)DS(2) score can be useful for predicting post-stroke pneumonia and for routine monitoring of high-risk elderly stroke patients in the clinical setting.

[A follow-up study on cognitive changes of mild cognitive impairment in the elderly].

OBJECTIVE: To observe and assess the cognitive changes of mild cognitive impairment (MCI) in the elderly. METHODS: A cohort study design was conducted among 47 patients with MCI and 21 control selected from the same convalescent camp, Montreal cognitive assessment (MoCA), mini mental state examination (MMSE) and clock drawing test (CDT) were performed to all subjects at the onset of study and 12 months later. RESULTS: The score of MMSE, CDT, MoCA and its subitems including visuospatial skill and delayed recall of MCI group were lower than the baseline after 12 months, with significantly decline in the score of MoCA (P = 0.041) and delay recall (P = 0.003). There was no obvious difference in the score of control between the baseline and that after 12 months. CONCLUSION: The decline of delayed recall occurred early and significantly, which may be a predictor in the conversion of mild cognitive impairment to dementia.

[Association analysis between SNPs of the growth hormone receptor gene and growth traits in arctic fox].

Using single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing, single nucleotide polymorphisms (SNPs) of growth hormone receptor (GHR) gene were detected in an arctic fox population. Correlation analysis between GHR polymorphisms and growth traits were carried out using the appropriate model. Four SNPs, G3A in the 5'UTR, C99T in the first exon, T59C and G65A in the fifth exon were identified on the arctic fox GHR gene. The G3A and C99T polymorphisms of GHR were associated with female fox body weight (Pamp;0.05) and the T59C and G65A polymorphisms of GHR were associated with male fox body weight (Pamp;0.05) and the skin length of the female fox (Pamp;0.01). Therefore, marker assistant selection on body weight and skin length of arctic foxes using these SNPs can be applied to get big and high quality arctic foxes.

Bioinformatic analysis of microRNA biogenesis and function related proteins in eleven animal genomes.

MicroRNAs are approximately 22 nt long small non-coding RNAs that play important regulatory roles in eukaryotes. The biogenesis and functional processes of microRNAs require the participation of many proteins, of which, the well studied ones are Dicer, Drosha, Argonaute and Exportin 5. To systematically study these four protein families, we screened 11 animal genomes to search for genes encoding above mentioned proteins, and identified some new members for each family. Domain analysis results revealed that most proteins within the same family share identical or similar domains. Alternative spliced transcript variants were found for some proteins. We also examined the expression patterns of these proteins in different human tissues and identified other proteins that could potentially interact with these proteins. These findings provided systematic information on the four key proteins involved in microRNA biogenesis and functional pathways in animals, and will shed light on further functional studies of these proteins.

Establishment and characterization of a fibroblast cell line derived from Texel sheep.

A Texel sheep ear marginal tissue fibroblast cell line (named TSF19) was successfully established by using a primary explant technique and cell cryoconservation technology. TSF19 cells were adherent, with a population doubling time of 24.9 h. Chromosome analysis showed that >90% of cells were diploid prior to cell passage 4. Isoenzyme analyses of lactate dehydrogenase and malate dehydrogenase showed that the TSF19 cells had no cross-contamination with other species. Tests for cell line contamination with bacteria, fungi, or mycoplasmas were also negative. Plasmids encoding the fluorescent proteins pEGFP-N3, pECFP-N1, pDsRed1-N1, and pEYFP-N1 were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between 21.8% and 46.5%. This newly established cell line will not only preserve the genetic resources of the important Texel sheep at the cell level but will also provide a valuable resource for genomic, postgenomic, somatic cloning research.

Establishment and characterization of a fibroblast cell line from the Mongolian horse.

A fibroblast line was successfully established from Mongolian horse ear marginal tissue by using a primary explant technique and cell cryogenic preservation technology. Biological analysis showed the following: The cells were adherent and exhibited density-dependent inhibition of proliferation; assays of microbial contamination from bacteria, fungi, and mycoplasma were negative; the population doubling time of the cells was 33.9 h; and a 2n chromosome number of 64 at a frequency higher than 80%. A lack of cross-contamination of this cell line with other species was confirmed by isoenzyme analysis of lactic and malic dehydrogenases. In order to study exogenous gene expression, four fluorescent proteins, pEGFP-N3, pEGFP-C1, pDsRed1-N1, and pEYFP-N1, were transfected into the cells. The corresponding fluorescence was distributed throughout the cytoplasm and nucleus 12 h after transfection. This cell line not only preserves the genetic resources of the Mongolian horse at the cellular level but also provides valuable materials for genomic, postgenomic, and somacloning research in this species.

Differentiation potential of bone marrow mesenchymal stem cells in duck.

The bone marrow mesenchymal stem cells (MSCs) are multipotent stem cells which can differentiate into mesenchymal cells in vitro. In this study, MSCs in duck were isolated from bone marrow by density gradient centrifuge separation, purified and expanded in the medium. The primary MSCs were expanded for passages. The different-passage MSCs were induced to differentiate into osteoblasts and neuron-like cells. Karyotype analysis indicated that MSCs kept diploid condition and the hereditary feature was stable. The different-passage MSCs expressed CD44, ICAM- and SSEA-4, but not CD34, CD45 and SSEA-when detected by immunofluorescence staining. There was no significant difference among the positive rates of passages 2, 6 and 8 (P > 0.05), but a significant difference existed among those of passages 2, 6, 8 and 11 (P < 0.05). After the osteogenic inducement was added, the induced different-passage MSCs expressed high-level alkaline phosphatase (ALP), and are positive for tetracycline staining, Alizarin Red staining and Von Kossa staining. After the neural inducement was added, about 70% cells exhibited typical neuron-like phenotype, the induced different-passage MSCs expressed Nestin, neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) when detected by immunofluorescence staining. There was no significant difference among the positive rates of passages 3, 4 and 6 (P>0.05), but a significant difference existed among those of passages 3, 4, 6 and 8 (P<0.05). These results suggest that MSCs in duck were capable of differentiating into osteoblasts and neuron-like cells in vitro.