Effects of inhibin silencing on INHβB, FSHβB, LHβB, GnRHR and β-glycan.

<p>The levels of INHβB, FSHβB, LHβB, GnRHR and β-glycan were quantified by real-time PCR and western-blotting among pshRNAi-2, pshRNAi-negative and PBS groups, respectively. <b>A:</b> The mRNA levels of INHβB, FSHβB, LHβB, GnRHR and β-glycan were examined by q-PCR. Compared with pshRNA-negative and PBS groups, the mRNA levels of INHβB, FSHβB, LHβB and GnRHR were up-regulated [P>0.05] in APCs transfected with pshRNAi-2. However,mRNA levels of INHβB and GnRHR were significantly up-regulated, whereas mRNA level of β-glycan was significantly down-regulated. B: The proteins levels of FSH, LH, ACVB, INHB, GnRHR and β-glycan were detected by western blotting. The results showed that FSH, LH, ACVB, INHB, GnRHR proteins were up-regulated by inhibin gene silencing. But the protein of β-glycan was down-regulated by inhibin silencing.</p

Analysis of Cell Cycle by Flow Cytometry in the anterior pituitary cells 48-2, pshRNA-negative and PBS [Mean ± SEM, n = 3].

<p>All results were evaluated by One-way ANOVA.</p><p>[*] indicates level of significance in columns [P<0.05].</p

Measurement of mouse pituitary cell apoptosis.

<p>There were significantly less apoptotic cells and more vital cells in the pShRNA-2 group compared to those of pShRNA-negative and PBS group respectively. [Means±SE, n = 3].</p><p>All results were evaluated by One-way ANOVA,</p><p>[*] P<0.05</p><p>[**] P<0.01 indicates level of significance in columns.</p

Geographical distribution of serum samples.

<p>Geographical distribution of serum samples.</p

Detection of inhibin α-subunit expression in the mouse anterior pituitary.

<p>A: Inhibin α-subunit expression in the mouse anterior pituitary gland using immunohistochemical methods. Immunohistochemical analysis of inhibin α subunit expression in the anterior pituitary gland by inhibin monoclonal antibody [A1] and PBS [A2], magnification is ×40. B: Inhibin α subunit expression in the cultured mouse anterior pituitary cells using indirect immunofluorescence. Indirect immunofluorescence analysis of inhibin α subunit expression in the anterior pituitary cells by inhibin monoclonal antibody [B1] and PBS [B2], magnification is ×40.</p

Inhα RNAi recombinant plasmids expression in the anterior pituitary cells.

<p>Three Inhα RNAi recombinant plasmids were transfected in anterior pituitary cells named as pshRNA-1, pshRNA-2, pshRNA-3 and pshRNA-negative, respectively for 24 h, 48 h or 72 h, the best efficient of which was selected for further investigation.</p

Concentration of INHB and ACVB in the culture medium released by the mouse anterior pituitary cells at the different times after transfection.

<p>A: Detection of the INHB concentration was done by RIA kit. Compared with pshRNAi-negative and PBS groups, the INHB concentrations was decreased significantly at 24 and 48 h after transfection with pshRNAi-2. B: Similarly, compared with pshRNAi-negative and PBS groups, the ACVB concentrations was increased significantly at 24 and 48 hours after transfection with pshRNAi-2.</p

Concentration of LH in the culture medium released by the mouse anterior pituitary cells at the different times after transfection.

<p>The results indicated that the LH concentrations in the group were not affected by inhibin gene.</p

Comparisons between BVDV Ab and nucleic acid detection by RT-PCR.

<p>Comparisons between BVDV Ab and nucleic acid detection by RT-PCR.</p

Prevalence Study and Genetic Typing of Bovine Viral Diarrhea Virus (BVDV) in Four Bovine Species in China

<div><p>To determine the nationwide status of persistent BVDV infection in different bovine species in China and compare different test methods, a total of 1379 serum samples from clinical healthy dairy cattle, beef cattle, yaks (<i>Bos grunniens</i>), and water buffalo (<i>Bubalus bubalis</i>) were collected in eight provinces of China from 2010 to 2013. The samples were analyzed using commercial antibody (Ab) and antigen (Ag) detection kits, and RT-PCR based on the 5’-UTR and Npro gene sequencing. Results showed that the overall positive rates for BVDV Ab, Ag and RT-PCR detection were 58.09% (801/1379), 1.39% (14/1010), and 22.64% (146/645), respectively, while the individual positive rates varied among regions, species, and farms. The average Ab-positive rates for dairy cattle, beef cattle, yaks, and water buffalo were 89.49% (298/333), 63.27% (248/392), 45.38% (236/520), and 14.18% (19/134), respectively, while the Ag-positive rates were 0.00% (0/116), 0.77% (3/392), 0.82% (3/368), and 5.97% (8/134), respectively, and the nucleic acid-positive rates detected by RT-PCR were 32.06% (42/131), 13.00% (26/200), 28.89% (52/180), and 19.40% (26/134), respectively. In addition, the RT-PCR products were sequenced and 124 5’-UTR sequences were obtained. Phylogenetic analysis of the 5’-UTR sequences indicated that all of the 124 BVDV-positive samples were BVDV-1 and subtyped into either BVDV-1b (33.06%), BVDV-1m (49.19%), or a new cluster, designated as BVDV-1u (17.74%). Phylogenetic analysis based on Npro sequences confirmed this novel subtype. In conclusion, this study revealed the prevalence of BVDV-1 in bovine species in China and the dominant subtypes. The high proportion of bovines with detectable viral nucleic acids in the sera, even in the presence of high Ab levels, revealed a serious threat to bovine health.</p></div