Comparative proteomic analysis of seminal plasma exosomes in buffalo with high and low sperm motility.

BACKGROUND: Exosomes are nanosized membranous vesicles secreted by various types of cells, which facilitate intercellular communication by transporting bioactive compounds. Exosomes are abundant in biological fluids including semen, and their protein composition and the potential of seminal plasma exosomes (SPEs) as fertility biomarkers were elucidated in humans, however, little information is available regarding buffalo (Bubalus bubalis). Here, we examined protein correlation between spermatozoa, seminal plasma (SP), and SPEs, and we compared and analyzed protein differences between high-motility (H-motility) and low-motility (L-motility) SPEs in buffalo. RESULTS: SPEs were concentrated and purified by ultracentrifugation combined with sucrose density gradient centrifugation, followed by verification using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Protein composition in spermatozoa, SP and SPEs, and protein difference in H- and L-motility SPEs were identified by LC-MS/MS proteomic analysis and were functionally analyzed through comprehensive bioinformatics. Many SPEs proteins originated from spermatozoa and SP, and nearly one third were also present in spermatozoa and SP. A series of proteins associated with reproductive processes including sperm capacitation, spermatid differentiation, fertilization, sperm-egg recognition, membrane fusion, and acrosome reaction were integrated in a functional network. Comparative proteomic analyses showed 119 down-regulated and 41 up-regulated proteins in L-motility SPEs, compared with H-motility SPEs. Gene Ontology (GO) enrichment of differentially expressed proteins (DEPs) showed that most differential proteins were located in sperm and vesicles, with activities of hydrolase and metalloproteinase, and were involved in sperm-egg recognition, fertilization, single fertilization, and sperm-zona pellucida binding processes, etc. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differential proteins were mainly involved in the PPRP signaling pathway, calcium signaling pathway, and cAMP signaling pathway, among others. Furthermore, 6 proteins associated with reproduction were validated by parallel reaction monitoring analysis. CONCLUSION: This study provides a comprehensive description of the seminal plasma exosome proteome and may be of use for further screening of biomarkers associated with male infertility.

Comparison of the effects of buffalo LIF and mouse LIF on the in vitro culture of buffalo spermatogonia.

Leukemia inhibitory factor (LIF) is an important growth factor that supports the culture and maintenance of spermatogonial stem cells (SSCs) by suppressing spontaneous differentiation. Different LIF sequences may lead to differences in function. The protein sequences of buffalo LIF and mouse LIF differed by 65.5% according to MEGA software analysis. The PB-LIF-GFP-Puro vector was constructed, and the CHO-K1 cell line was established. The final LIF protein concentration in the CHO-K1 cell culture medium was approximately 4.268 ng/mL. Here, we report that buffalo LIF effectively maintains the self-renewal of buffalo spermatogonia during culture. Buffalo spermatogonia were cultured in conditioned medium containing no LIF (0 ng/mL), mouse LIF (1 ng/mL), mouse LIF (10 ng/mL), or buffalo LIF (1 ng/mL). Furthermore, the effects of mouse LIF and buffalo LIF culture on the maintenance of buffalo spermatogonia were determined by analyzing cell colony formation, quantitative real-time polymerase chain reaction, cell immunofluorescence, and cell counting. The buffalo LIF (1 ng/mL) group showed similar maintenance of the proliferation of buffalo spermatogonia to that in the mouse LIF (10 ng/mL) group. These results demonstrated that the proliferation of buffalo spermatogonia can be maintained in vitro by adding a low dose of buffalo LIF. This study provides a foundation for the further optimization of in vitro buffalo SSC culture systems.

Predictors of Kidney Outcomes of Anti-Glomerular Basement Membrane Disease in a Large Chinese Cohort.

INTRODUCTION: Anti-glomerular basement membrane (GBM) disease is a rare but the most aggressive form of glomerulonephritis. To dissect the prognostic factors, we retrospectively analyzed the clinical features of a large cohort and compared the clinical features and prognosis during decades. METHODS: Data on clinical manifestation, treatment, and prognosis were collected. Cox models and receiver operating characteristic (ROC) curve were used to investigate the predictors for outcomes. The Kaplan-Meier curve and log-rank test were used to compare kidney and patient survival. RESULTS: A total of 448 patients were enrolled. Patient survival and kidney survival at 1 year was 69.4% and 37.7%, respectively. During the past 3 decades, mortality at 3 months and 1 year significantly dropped from 37.5% and 57.1% in 1991-2000 to 2.8% and 6.9% in 2011-2020 (p < 0.001), respectively; kidney prognosis showed a tendency of improvement as well. Serum creatinine (Scr) on diagnosis (HR, 1.16; 95% CI, 1.05-1.29) and crescent percentage (HR, 1.73; 95% CI, 1.34-2.24) were independent predictors for end-stage kidney disease. ROC curve showed that the optimal cutoff point of Scr on diagnosis for prediction of dialysis dependency at 1 year was 536.4 µmol/L (sensitivity 88.3% and specificity 80.8%). Antineutrophil cytoplasmic antibodies (ANCAs) positivity (HR, 4.43; 95% CI, 1.72-11.38) was a predictor for mortality. Plasma exchange was associated with a better patient prognosis (HR, 0.40; 95% CI 0.16-0.95). CONCLUSION: Scr on diagnosis and percentage of crescents were predictors for kidney outcomes. Positive ANCA was a predictor for mortality. Overall patient prognosis of anti-GBM disease was improved during the past 3 decades.

Congenital sensorineural hearing loss as the initial presentation of PTPN11-associated Noonan syndrome with multiple lentigines or Noonan syndrome: clinical features and underlying mechanisms.

BACKGROUND: Germline variants in PTPN11 are the primary cause of Noonan syndrome with multiple lentigines (NSML) and Noonan syndrome (NS), which share common skin and facial symptoms, cardiac anomalies and retardation of growth. Hearing loss is considered an infrequent feature in patients with NSML/NS. However, in our cohort, we identified a group of patients with PTPN11 pathogenic variants that were primarily manifested in congenital sensorineural hearing loss (SNHL). This study evaluated the incidence of PTPN11-related NSML or NS in patients with congenital SNHL and explored the expression of PTPN11 and the underlying mechanisms in the auditory system. METHODS: A total of 1502 patients with congenital SNHL were enrolled. Detailed phenotype-genotype correlations were analysed in patients with PTPN11 variants. Immunolabelling of Ptpn11 was performed in P35 mice. Zebrafish with Ptpn11 knockdown/mutant overexpression were constructed to further explore mechanism underlying the phenotypes. RESULTS: Ten NSML/NS probands were diagnosed via the identification of pathogenic variants of PTPN11, which accounted for ~0.67% of the congenital SNHL cases. In mice cochlea, Shp2, which is encoded by Ptpn11, is distributed in the spiral ganglion neurons, hair cells and supporting cells of the inner ear. In zebrafish, knockdown of ptpn11a and overexpression of mutant PTPN11 were associated with a significant decrease in hair cells and supporting cells. We concluded that congenital SNHL could be a major symptom in PTPN11-associated NSML or NS. Other features may be mild, especially in children. CONCLUSION: Screening for PTPN11 in patients with congenital hearing loss and variant-based diagnoses are recommended.

Maturation of buffalo oocytes in vitro with acetyl-L-carnitine improves cryotolerance due to changes in mitochondrial function and the membrane lipid profile.

The effects of acetyl-l-carnitine (ALC) supplementation during IVM on subsequently vitrified buffalo oocytes were evaluated, followed by determination of the mitochondrial DNA copy number, measurement of mitochondrial membrane potential (MMP) and identification of the lipid profile of oocyte membranes as markers of oocyte quality after vitrification. Supplementation with ALC during IVM significantly improved the rates of oocyte cleavage and morula and blastocyst formation, and increased MMP after vitrification compared with unsupplemented vitrified oocytes (P<0.05). Using a bidirectional orthogonal projection to latent structures discriminant analysis based on positive ion matrix-assisted laser desorption ionisation time-of-flight mass spectrometry data, five phospholipid ions (m/z 728.7 (phosphatidylcholine (PC) 32:3), 746.9 (PC 32:5), 760.6 (PC 34:1), 768.8 (PC P-36:3) and 782.6 (PC 36:4); P<0.05) were identified as significantly more abundant in fresh oocytes than in unsupplemented vitrified oocytes. Meanwhile, three phospholipid ions (m/z 734.6 (PC 32:0), 760.6 (PC 34:1), and 782.6 (PC 36:4); P<0.05) were more abundant in ALC-supplemented vitrified oocytes than in unsupplemented vitrified oocytes. Therefore, supplementation with ALC during IVM may improve buffalo oocyte quality after vitrification by enhancing mitochondrial function and altering the phospholipid composition of vitrified oocyte membranes.

Methylation characteristics and developmental potential of Guangxi Bama minipig (Sus scrofa domestica) cloned embryos from donor cells treated with trichostatin A and 5-aza-2'-deoxycytidine.

Summary Reprogramming of DNA methylation in somatic cell nuclear transfer (SCNT) embryos is incomplete, and aberrant DNA methylation patterns are related to the inefficiency of SCNT. To facilitate nuclear reprogramming, this study investigated the effect of treating Guangxi Bama minipig donor cells with trichostatin A (TSA), 5-aza-2'-deoxycytine (5-aza-dC), or combination of TSA and 5-aza-dC prior to nuclear transfer. Analyses showed that there were no major changes in cell-cycle status among all groups. We monitored the transcription of DNMT1, DNMT3a, HDAC1 and IGF2 genes in donor cells. Transcription levels of HDAC1 were decreased significantly after treatment with a combination of TSA and 5-aza-dC, along with a significantly increased level of IGF2 (P < 0.05). Although treatment of donor cells with either TSA or 5-aza-dC alone resulted in non-significant effects in blastocyst formation rate and DNA methylation levels, a combination of TSA and 5-aza-dC significantly improved the development rates of minipig SCNT embryos to blastocyst (25.6% vs. 16.0%, P < 0.05). This change was accompanied by decreased levels of DNA methylation in somatic cells and blastocyst (P < 0.05). Thus in combination with TSA, lower concentrations of 5-aza-dC may produce a potent demethylating activity, and lead to the significantly enhanced blastocyst development percentage of Bama minipig SCNT embryos.

Developmental potential of buffalo embryos cultured in serum free culture system.

The presence of serum in embryo culture medium has been implicated for increased embryo's sensitivity to cryopreservation, compromised viability, abnormal embryo and fetal development. Hence, designing a serum free culture system is indispensable. The present study aims to compare the efficiency of the serum and granulosa cells monolayer free commercial culture system (SFCS) with the conventional serum supplemented co-culture system (SSCS) and optimized culture system (OCS). Generally, SFCS is designed explicitly for bovine oocyte maturation and embryo culture (SF-IVM and SF-IVC), and SSCS (based on M199, SS-IVM, and SS-IVC) is utilized for buffalo in vitro embryo production. However, OCS is a newly designed culture system in which oocyte maturation is performed in serum supplemented maturation medium, and the subsequent embryos are co-cultured with granulosa cells in serum free culture medium. To evaluate the effect of serum on buffalo embryo production, buffalo oocytes, and their subsequent embryos were cultured in SSCS, SFCS, and OCS, simultaneously. The percentage of cleaved embryos cultured in SSCS and OCS was approximately 4% higher as compared to SFCS. However, OCS significantly showed the maximum proportion of embryos that developed to the blastocyst stage (7d) and hatched (6d) as compared to the SFCS and SSCS. Additionally, OCS promoted the expression of developmentally important genes (BCL2-L1 and VEGF-A), cell number, and cryo-survival ability of blastocysts in comparison with SSCS. Taken together, OCS is more suitable for the oocyte maturation and culture of buffalo embryos. However, to design the serum free culture system, it is recommended to find suitable serum alternatives for in vitro oocyte maturation.

Effects of different culture systems on the culture of prepuberal buffalo (Bubalus bubalis) spermatogonial stem cell-like cells in vitro.

Currently, the systems for culturing buffalo spermatogonial stem cells (SSCs) in vitro are varied, and their effects are still inconclusive. In this study, we compared the effects of culture systems with undefined (foetal bovine serum) and defined (KnockOut Serum Replacement) materials on the in vitro culture of buffalo SSC-like cells. Significantly more DDX4- and UCHL1-positive cells (cultured for 2 days at passage 2) were observed in the defined materials culture system than in the undefined materials system (p < 0.01), and these cells were maintained for a longer period than those in the culture system with undefined materials (10 days vs. 6 days). Furthermore, NANOS2 (p < 0.05), DDX4 (p < 0.01) and UCHL1 (p < 0.05) were expressed at significantly higher levels in the culture system with defined materials than in that with undefined materials. Induction with retinoic acid was used to verify that the cultured cells maintained SSC characteristics, revealing an SCP3⁺ subset in the cells cultured in the defined materials system. The expression levels of Stra8 (p < 0.05) and Rec8 (p < 0.01) were significantly increased, and the expression levels of ZBTB16 (p < 0.01) and DDX4 (p < 0.05) were significantly decreased. These findings provided a clearer research platform for exploring the mechanism of buffalo SSCs in vitro.

[Intervention of auricular point sticking on perioperative psychological stress in patients with anorectal diseases].

OBJECTIVE: To explore and quantify the intervention effect of auricular point sticking on perioperative psychological stress in patients with anorectal diseases. METHODS: Eighty patients who underwent anorectal surgery were randomly divided into an observation group (40 cases) and a control group (40 cases). The routine preoperative guidance, preoperative visits, and informed of the postoperative condition were received in the control group. On the basis of the treatment in the control group, auricular point sticking was immediately applied at Shenmen (TF4), Shen (CO10), Wei (CO4), Gan (CO12), Pi (CO13), Pizhixia (AT4), E (AT1), Nie (AT2) and Zhen (AT3) in the observation group.The patients were pressed by themselves, 3 to 5 min per point each time, 5 times a day, and the contralateral auricular points were replaced every 2 or 3 days until 1 week after surgery. The Hamilton anxiety scale (HAMA), Hamilton depression scale (HAMD), and Pittsburgh sleep quality index (PSQI) scores were compared between the two groups before and 7 days after surgery. RESULTS: There was no significant difference in the total HAMA scores between after and before surgery in the observation group (P>0.05). The total HAMA score in the control group was higher than that before surgery (P<0.05). The total HAMA score in the observation group after surgery was lower than that in the control group (P<0.05). There was no significant difference in the total HAMD scores between the two groups before and after surgery (P<0.05). There was no significant difference in the total HAMD scores between the two groups after the surgery (P>0.05). The scores of somatic anxiety factor in the two groups were higher than those before surgery (P<0.05). The scores of somatic anxiety factor in the observation group were lower than those in the control group (P<0.05). The scores of psychotic anxiety factors in the two groups were lower than those before surgery (P<0.05). There was no significant difference in the score of psychotic anxiety factors between the two groups (P>0.05). The total score of PSQI in the two groups was lower than that before surgery (P<0.05), and the total score of PSQI in the observation group was lower than that in the control group (P<0.05). CONCLUSION: Auricular point sticking can effectively improve some psychological stress problems during perioperative period in patients with anorectal diseases.

Association of liver enzymes levels with fasting plasma glucose levels in Southern China: a cross-sectional study.

OBJECTIVE: According to several studies, liver enzymes levels are associated with fasting plasma glucose (FPG) levels. However, the association stratified by body mass index (BMI) remains to be elucidated, especially in Southern China. Therefore, the aim of this study was to investigate the correlation between liver enzymes levels and FPG levels stratified by BMI in Southern China. DESIGN: Cross-sectional study. PARTICIPANTS AND SETTING: 3056 individuals participated in real-time interviews and blood tests in Southern China. Participants were divided into three groups (underweight, normal weight and overweight or obesity) based on BMI cut-offs. MAIN OUTCOME MEASURED: Partial correlation analysis was performed to investigate the relationship between FPG levels and liver tests. Multivariate logistic regression analyses were applied to calculate the adjusted ORs for FPG levels based on liver enzymes levels. RESULTS: There was no association between liver enzymes and FPG either in the underweight group or in the normal weight group; however, a significant correlation was observed in the overweight or obesity group (alanine transaminase (ALT), p<0.01; aspartate aminotransferase (AST), p<0.05). After adjusting for confounding factors, the highest tertiles of ALT still remained significantly positively related to FPG levels in the overweight or obesity group, with an OR of 2.205 (95% CI 1.442 to 3.371) for the 5.56≤FPG<7.00 mmol/L vs the FPG<5.56 mmol/L group and with an OR of 2.297 (95% CI 1.017 to 5.187) for the FPG≥7.00 mmol/L vs the FPG<5.56 mmol/L group, but this correlation was not found for AST. CONCLUSIONS: The association of liver enzymes levels with FPG levels differed based on different BMI cut-offs. ALT levels were significantly positively associated with FPG levels in the overweight or obesity group, but not in the other two groups; AST levels were not associated with FPG levels in any group.

Hexagonal VO2 particles: synthesis, mechanism and thermochromic properties.

Monoclinic vanadium dioxide VO2 (M) with hexagonal structure is synthesized by hydrothermal method, and the phase evolution is evidenced. Interestingly, the hexagonal morphology comes into being as a result of the low-energy coherent interfaces, (211̄)1//(21̄1̄)2 and (21̄1̄)1//(020)2. The size of hexagonal particles is well controlled by changing the concentration of precursor solutions. Hexagonal particles exhibit excellent thermochromic properties with a narrow hysteresis of 5.9 °C and high stability. In addition, the phase transition temperature can be substantially reduced down to 28 °C by simply W doping.

Treatment with acetyl-l-carnitine during in vitro maturation of buffalo oocytes improves oocyte quality and subsequent embryonic development.

Oocyte quality is one of the important factors in female fertility, in vitro maturation (IVM), and subsequent embryonic development. In the present study, we assessed whether acetyl-l-carnitine (ALC) supplementation during in vitro maturation of buffalo oocytes could improve oocyte quality and subsequent embryonic development. To determine the optimal level of ALC supplementation, we matured cumulus-oocyte complexes in maturation medium supplemented with 0, 2.5, and 5 mM ALC. The oocytes with a polar body were selected for parthenogenetic activation (PA) and in vitro fertilization (IVF). We found that oocytes matured in 2.5 mM ALC had significantly higher PA blastocyst rate (P < 0.05) and blastocyst cell number than those of unsupplemented oocytes (P < 0.05) and a significantly higher IVF blastocyst rate than that of oocytes matured in 5 mM ALC (P < 0.05). In all further experiments, we supplemented the maturation medium with 2.5 mM ALC. We then tested whether ALC supplementation could improve various markers of oocytes and cumulus cells. We compared cell proliferation; concentrations of reactive oxygen species (ROS), intracellular ATP, estradiol, and progesterone; mitochondrial distribution; mitochondrial DNA copy number (mtDNA); and expression levels of four genes encoding oocyte-derived factors (GDF9, BMP15) and steroid hormones (StAR, P450scc) between the supplemented and unsupplemented oocytes and cumulus cells. Cumulus cells matured with ALC supplementation were more prolific than those matured without ALC supplementation (P < 0.05). Oocytes treated with ALC had lower concentrations of intracellular ROS (P < 0.05) and a higher rate of diffuse mitochondrial distributions (P < 0.05) than those of untreated oocytes. Additionally, the mtDNA was higher in the ALC-treated oocytes (P < 0.05) and cumulus cells (P < 0.05) than that in the untreated cells. The ALC-treated maturation medium had a higher postmaturation concentration of estradiol than that of the untreated medium (P < 0.05). Finally, the gene expression levels of P450scc and GDF9 were greater in ALC-treated oocytes and cumulus cells than those in untreated cells (P < 0.05). Therefore, in buffalo, our results suggest that ALC affects mitochondrial function, regulates oocyte-derived paracrine factors, and increases the production of steroid hormones, leading to increased quality of matured oocytes and improved embryonic development in vitro.

Trial to Incentivise Adherence for Diabetes (TRIAD): study protocol for a randomised controlled trial.

BACKGROUND: Many people with diabetes have suboptimal glycaemic control due to not being adherent to their treatment regimen. Behavioural economic theory suggests that the lack of adherence results from the disconnect between the timing of when costs and benefits accrue. One strategy to address this discontinuity is to offer patients a near-term benefit, such as a financial reward. Whereas there is evidence that rewards can improve treatment adherence and sometimes health outcomes, further research is needed to determine whether rewards are more effective when targeting processes or intermediary health outcomes. In the Trial to Incentivise Adherence for Diabetes (TRIAD) we test whether adding financial incentives to usual care can improve HbA1c levels among people with diabetes and whether the financial incentives work better when targeting processes (adherence to blood glucose testing, medication, and daily physical activity) or the primary intermediary health outcome of self-monitored blood glucose within an acceptable range. METHODS/DESIGN: TRIAD is a randomised, controlled, open-label, single-centre superiority trial with three parallel arms. A total of 240 patients with suboptimally controlled diabetes (HbA1c ≥ 8%) from a polyclinic in Singapore are block-randomised (blocking factor: current vs. new glucometer users) into three arms, namely (1) usual care (UC) only, (2) UC with process incentive and (3) UC with outcome incentive, in a 2:3:3 ratio. Masking the arm allocation will be precluded by the behavioural nature of the intervention but blocking size will not be disclosed to protect concealment. The primary outcome (change in HbA1c level at month 6) will be measured by a laboratory that is independent from the study team. Secondary outcomes (at month 6) include the number of blood glucose testing days, glucose readings within the normal range (between 4 to 7 mmol/L), medication-adherent days, physically active days, and average incentives earned and time spent administrating the incentives. DISCUSSION: This study will provide evidence on whether financial incentives can cost-effectively improve glycaemic control. It will also provide evidence on the benefit incidence of interventions involving financial incentives. By comparing process to outcome incentives, this study will inform the design of future incentive strategies in chronic disease management and beyond. TRIAL REGISTRATION: ClinicalTrials.gov registry, ID: NCT02224417 . Registered on 22 August 2014.

Nanos2 is a molecular marker of inchoate buffalo spermatogonia.

Nanos2 belongs to the Nanos gene-coding family and is an important RNA-binding protein that has been shown to have essential roles in male germline stem cells development and self-renewal in mouse. However, little is known about Nanos2 in inchoate buffalo spermatogonia. Here, rapid-amplification of cDNA ends (RACE) was used to obtain the full-length buffalo Nanos2 sequence and bioinformatic analysis revealed a highly conserved Nanos2 sequence between buffalo and other mammalian species. Although Nanos2 was expressed in various tissues, the highest mRNA expression levels were found in testes tissue. Moreover, Nanos2 mRNA was abundant in fetal and pre-puberal testes but markedly decreased in the testes of adults. At the protein level, immunohistochemistry in pre-puberal testes revealed a pattern of NANOS2 expression similar to that for the undifferentiated type A spermatogonia marker PGP9.5. Furthermore, NANOS2 expression was low in adult testes and restricted to elongating spermatids. Altogether, our data suggest that Nanos2 is a potential preliminary molecular marker of inchoate buffalo spermatogonia, and may play an important role in buffalo spermatogonial stem cells (SSCs) development and self-renewal, as has been observed in other model animals.

Flow cytometric and near-infrared Raman spectroscopic investigation of quality in stained, sorted, and frozen-thawed buffalo sperm.

Flow cytometry and Laser Tweezers Raman spectroscopy have been used to investigate Nili-Ravi buffalo (Bubalus bubalis) sperm from different samples (fresh, stained, sorted and frozen-thawed) of the flow-sorting process to optimize sperm sex sorting procedures. During the sorting and freezing-thawing processes, the two detection methods both indicated there were differences in mitochondrial activity and membrane integrity. Moreover, a dispersive-type NIR (Near Infrared Reflection) use of the Raman system resulted in the ability to detect a variety of sperm components, including relative DNA, lipid, carbohydrates and protein contents. The use of the Raman system allowed for PCA (principal components analysis) and DFA (discriminant function analysis) of fresh, stained, sorted and frozen-thawed sperm. The methodology, therefore, allows for distinguishing sperm from different samples (fresh, stained, sorted and frozen-thawed), and demonstrated the great discriminative power of ANN (artificial neural network) classification models for the differentiating sperm from different phases of the flow-sorting process. In conclusion, the damage induced by sperm sorting and freezing-thawing procedures can be quantified, and in the present research it is demonstrated that Raman spectroscopy is a valuable technology for assessing sperm quality.

Successful cloning of an adult breeding boar from the novel Chinese Guike No. 1 swine specialized strain.

Somatic cloning, also known as somatic cell nuclear transfer (SCNT), is a promising technology which has been expected to rapidly extend the population of elaborately selected breeding boars with superior production performance. Chinese Guike No. 1 pig breed is a novel swine specialized strain incorporated with the pedigree background of Duroc and Chinese Luchuan pig breeds, thus inherits an excellent production performance. The present study was conducted to establish somatic cloning procedures of adult breeding boars from the Chinese Guike No. 1 specialized strain. Ear skin fibroblasts were first isolated from a three-year-old Chinese Guike No. 1 breeding boar, and following that, used as donor cell to produce nuclear transfer embryos. Such cloned embryos showed full in vitro development and with the blastocyst formation rate of 18.4 % (37/201, three independent replicates). Finally, after transferring of 1187 nuclear transfer derived embryos to four surrogate recipients, six live piglets with normal health and development were produced. The overall cloning efficiency was 0.5 % and the clonal provenance of such SCNT derived piglets was confirmed by DNA microsatellite analysis. All of the cloned piglets were clinically healthy and had a normal weight at 1 month of age. Collectively, the first successful cloning of an adult Chinese Guike No. 1 breeding boar may lay the foundation for future improving the pig production industry.

Promoter structures and differential responses to viral and non-viral inducers of chicken melanoma differentiation-associated gene 5.

Melanoma differentiation-associated gene 5 (MDA5) is a member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) family and plays a pivotal role in the anti-viral innate immune response. As RIG-I is absent in chickens, MDA5 is hypothesized to be important in detecting viral nucleic acids in the cytoplasm. However, the molecular mechanism of the regulation of chicken MDA5 (chMDA5) expression has yet to be fully elucidated. With this in mind, a ∼2.5kb chMDA5 gene promoter region was examined and PCR amplified to assess its role in immune response. A chMDA5 promoter reporter plasmid (piggybac-MDA5-DsRed) was constructed and transfected into DF-1 cells to establish a Piggybac-MDA5-DsRed cell line. The MDA5 promoter activity was extremely low under basal condition, but was dramatically increased when cells were stimulated with polyinosinic: polycytidylic acid (poly I:C), interferon beta (IFN-ß) or Infectious Bursal Disease Virus (IBDV). The DsRed mRNA level represented the promoter activity and was remarkably increased, which matched the expression of endogenous MDA5. However, Infectious Bronchitis Virus (IBV) and Newcastle disease virus (NDV) failed to increase the MDA5 promoter activity and the expression of endogenous MDA5. The results indicated that the promoter and the Piggybac-MDA5-DsRed cell line could be utilized to determine whether a ligand regulates MDA5 expression. For the first time, this study provides a tool for testing chMDA5 expression and regulation.

Establishment and biological characteristics comparison of Chinese swamp buffalo (Bubalus bubalis) fibroblast cell lines.

To establish fibroblast cell lines from different tissues and to compare the biological characteristics of those cell lines, five fibroblast cell lines derived from Chinese swamp buffalo (Bubalus bubalis) were selected for comparative assays. Cell style and survival rate (before cryogenic preservation and after recovery) were tested, and karyotype, patterns of isoenzymes of lactic dehydrogenase, malic dehydrogenase, and cell cycle were analyzed. These cell lines had a healthy morphology with a typical spindle shape, and assessment of cell style showed these cells to be very pure fibroblasts. Cell growth curves showed a typical "S" shape. Results of microorganism contamination assays were negative, and isoenzyme analysis showed no cross-contamination. The number of chromosomes (2n) of swamp buffalo is 48. Between 28% and 46% of the cells were 2n, and cell apoptosis was not pronounced at 20th generation. Results showed that skin fibroblasts were more adaptable to tissue culture conditions than the ones from kidneys and ear margin, and they are more suitable for cellular manipulation in Chinese swamp buffalo.