Effect of nitric oxide on Sertoli cell microtubule of piglets

To illustrate the effect of nitric oxide (NO) on the microtubules of Sertoli cells (SC), SCs of piglets were treated with sodium nitroprusside (SNP). Changes in cell viability, anti-oxidant activity, enzyme activity and p38 mutagen-activated protein kinase (p38MAPK) activation were detected. The results were as follows. A low concentration of NO can keep SC microtubule and cell viability normal, and a high concentration of NO could increase p38MAPK activation, decrease anti-oxidant activity and transferrin secretion, and destroy the structure and distribution of the microtubules. The results suggest that SNP treatment results in an increase in NO in SCs and decreased cell anti-oxidant activity. The high concentration of NO destroys cell microtubules by activating p38MAPK.

Purification and antibacterial activity of recombinant human lactoferrin in milk of transgenic mice

To verify its antibacterial activity, recombinant human lactoferrin (rhLF) was extracted from the milk of transgenic mice (Mus musculus) (PCL25and AP) by gel filtration chromatography and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and enzyme-linked immunosorbent assay (ELISA). In addition, its bacteriostatic properties were tested using the agar disc diffusion method. ELISA analysis showed that the concentration of rhLF in the milk of transgenic mice ranged from 7 to 8 mg/ml, and the recombinant protein expressed in the milk had the same molecular weight as the native protein (~78 kDa), indicating that the rhLFs had a strong antibacterial activity onEscherichia coliandSalmonella.

Isolation, mapping and application of a repetitive DNA sequence in wheat (Triticum aestivum) A and B genomes

Simple sequence repeat (SSR) analysis was performed on five Secale species, four Triticum species and a Triticale line Fenzhi-1 using 102 pairs of microsatellite primers. A 387-bp specific DNA fragment FZ387 (GenBank accession no. EF179137) was obtained from the Triticale Fenzhi-1 with primer Xgwm614, without amplification in Secale. NCBI BLAST revealed that this FZ387 sequence had 94% and 95% similarity to part of the Gypsy Ty3-LTR retrotransposon Fatima in Triticum monoccocum (AY485644) and Triticum turgidum (AY494981), respectively. A pair of specific polymerase chain reaction (PCR) primers, FaF and FaR, was designed based on the conserved region of this FZ387 sequence. The amplification of primer pair Xgwm614F and FaR revealed that a specific 350-bp band (designation as A350) was obtained from the species containing A chromosomes. Furthermore, PCR on Langdon Chinese Spring substitution lines was performed, and the results found that this segment was located on both long and short arms of all A chromosomes. However, the amplification of primer pair FaF and Xgwm614R gave rise to a specific DNA band of about 350 bp (designated AB350) from materials containing A and/or B chromosomes. The wild species of wheat and the relatives were amplified using the two pairs of primers, and revealed that only A350 and AB350 were found in Chinese Spring (CS). Sequence comparison and variation of SSR primers binding regions of FZ387 indicated that significant diversity might exist in the internal sequence of this Fatima-like element among triticeae genomes. Meanwhile, both A350 and AB350 can be used as molecular markers for the detection of A and AB genomes.

Collection and preservation of porcine polar bodies

Mature porcine oocytes containing first polar bodies (Pb I) were obtained by in vitro culture of follicle oocytes from ovaries obtained from a local abattoir, and zygotes with second polar bodies (Pb II) were grown after in vitro fertilization of the mature oocytes. Extrusion, biological activity and morphology of Pb I and Pb II were statistically analysed. Polar bodies were isolated and collected from oocytes by enzyme digestion or micromanipulation. Their vigour under different preservation conditions was analysed and evaluated using a Trypan blue staining method. The results showed that 66.7% of the oocytes extruded Pb I after 40 h of in vitro mature culture of oocytes, and 49.7% of the zygotes extruded Pb II 20 h after in vitro fertilization. The efficiency of isolation of Pb II by micromanipulation significantly exceeded that by enzyme digestion, the Pb I and Pb II isolated by micromanipulation presenting with good vigour and normal morphology (95.3% versus 58.9%). The survival rates of Pb I and Pb II were 63.3% and 93.1% for 4 h at 39°C, 85.0% and 72.9% for 40 h at 4°C, and over 95.0% and 84.6% for less than 7 days at −20°C. In comparison with the above preservation conditions for Pb I and Pb II, the results for cryopreservation were best, with rates of survival as high as 89.1% for Pb I and 87.9% for Pb II for preservation periods of over a month, and rates of normal morphology of 97.8% and 95.7%, respectively. The Pb I and Pb II could be isolated and preserved effectively, for use in further research on the recombination of oocytes and zygotes.

Expression, purification and identification of gibberellin-induced cysteine-rich protein ofGymanadenia conopsea

Primers bearing restriction enzyme sites forEcoR I andHind III were designed according to the known partial cDNA sequence for gibberellin-induced cysteine-rich protein and were then used to amplify the full-length open reading frame (ORF) and signal peptide-truncated fragment of thegcgasagene. Two fragments with lengths 319 and 238 bp were obtained and were further cloned into plasmid pET-32(a). Following transformation intoEscherichia coliBL21(DE3), the fusion proteins were observed to appear at ~26.0 and 25.2 kDa after induction from 1 mmol/l isopropyl-beta-D-thiogalactopyronoside (IPTG). The results of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) of an ultra-thin section revealed that the presence of signal peptide gave rise to the formation of an inclusion body located in the periplasmic space; however, the absence of signal peptide greatly enhanced the solubility of the target protein. The expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods.

Variation analysis of theVP1and3ABCgenes from yellow cattle isolates persistently infected byFoot-and-mouth disease virus

The potential relationship between the establishment ofFoot-and-mouth disease virus(FMDV) persistent infection and gene variation was identified by investigating the variation ofVP1and3ABCgenes from yellow cattle persistent infection isolates. Five yellow cattle were inoculated on their tongue with 1.0×104ID50/ml of FMDV O/Akesu/58 strain. After displaying clinical or subclinical signs, they probably became asymptomatic carriers. Oesophageal–pharyngeal fluids were collected monthly from the carriers with a probang and inoculated into a baby hamster kidney cell line (BHK-21); 12 FMDV isolates were obtained. TheVP1and3ABCgenes of the 12 isolates were then amplified by reverse-transcriptase polymerase chain reaction (RT-PCR). Cloning and sequencing revealed that the homology of theVP1nucleotide and amino-acid sequence of all the isolates was above 98%, with no base deletion or insertion. When compared with the O/Akesu/58 FMDV strain, the homology of theVP1nucleotide sequence of the isolates was only 85%, and that of the deduced amino-acid sequence only 90%.There were several nucleotide mutations in theVP1gene of the isolates, including 16 consistent nucleotide mutations, with only two of them leading to a change in amino acid (I56→T, A210→E). Moreover, it was found that four nucleotide points and three amino-acid points had transversions among all isolates. The3ABCgene had only 13 nucleotide transversions and five amino-acid mutations. It was presumed that persistent FMDV infection might have little connection with variation in theVP1and3ABCgenes, and was probably related to other structural protein gene and key factors.

The effects of diet protein levels on the growth, body composition and digestive enzyme activities of the Barbodes caldwelli juvenile

Seven isoenergetic semi-purified test diets containing graded levels of protein ranging from 20 to 50% were formulated using fish meal and casein as the protein sources. Test diets were fed to triplicate groups of Barbodes caldwelli juveniles with initial body weights of 1.26±0.02 g for eight weeks. The results indicated no significant effect of dietary protein levels on survival rate, relative weight of the viscera and relative weight of the liver in the juvenile fish. The weight gain and specific growth rate of the fish were found to be greater as dietary protein levels increased from 20 to 35%, but were not affected significantly as dietary protein level increased from 35 to 50%. Feed efficiencies did not differ significantly when fish were fed diets with protein levels from 30 to 50%, but were significantly higher than those of fish fed diets with protein levels of 20 and 25%. The protein efficiency ratio (PER) was negatively correlated with diet protein level (x) (PER=3.006−0.03251x, R=0.9366). There was no significant effect of dietary protein levels on carcass moisture, crude protein and ash. However, carcass lipid levels (L) decreased with an increase in dietary protein level (x) (L=8.2169−0.0458x, R=0.8551). There was no significant variation in hepatopancreas protease activity among the tests. Intestine protease activity and hepatopancreas amylase activity were increased to some extent, and then decreased as dietary protein levels continued to rise. The intestine amylase activity (IAA) of the juveniles was negatively correlated with dietary protein level (x) (IAA=84.625−0.9147x, R=0.8463). It was estimated that the suitable protein level for the B. caldwelli juvenile is 34% (the broken-line model was used to regress the relationship of the weight gain of the juvenile and dietary protein level).

Assessing genetic diversity in three wild Brachymystax lenok populations using AFLP markers

The genetic diversities of 72 individuals from three wild Lenok populations of Mudanjiang River (MD), Yalujiang River (YL) and Wusulijiang River (WSL) in the northeast of China were analysed using amplified fragment length polymorphism (AFLP) markers. The results showed that 541 polymorphic loci out of 559 were amplified by 12 primer pairs and the percentage of polymorphic loci was 96.78%. Shannon indices for the MD, YL and WSL populations were 0.3988±0.2913, 0.3254±0.3037, 0.2125±0.2862, respectively, and Nei's gene diversity indices were 0.2737±0.2062, 0.2229±0.2129, 0.1446±0.1985, respectively. The average total genetic diversity (Ht) was 0.3512±0.0.0208 and the average genetic diversity within populations (Hs) was 0.2137±0.0152. Among the three populations, the average genetic distance (Dst) was 0.1375 and the gene differentiation coefficient (Gst) was 0.3914. The genetic diversity was 60.85% within populations and 39.15% among populations. The gene flow index (Nm) was 0.7776. The analysis of molecular variance (AMOVA) indicated that the average fixation index (Fst) was 0.55336. The variance was 55.16% within populations and 44.84% among populations. The highest polymorphism ratio was in the MD group and the lowest in the WSL group.

Prokaryotic expression of nucleoprotein gene of Transmissible gastroenteritis virus and development of ELISA based on the expressed protein

The recombinant PET-N plasmid, which includes the N gene of the Transmissible gastroenteritis virus (TGEV), was transformed into Escherichia coli BL21 (DE3), and induced at 37°C with 1.0 mmol/l IPTG (isopropyl β-d-1-thiogalactopyranoside). An indirect enzyme-linked immunosorbent assay (ELISA) for detecting the TGEV nucleocapsid protein antibody was developed after the reactogenicity of the recombinant protein was demonstrated by Western blot. The operating conditions for the ELISA, an antigen concentration of 15 μg/ml, serum dilution of 1:40, blocking solution of 0.5% fetal bovine serum (FBS), serum sample incubation for 90 min, a concentration of horseradish peroxidase (HRP)-spa of 1:5000, incubated for 60 min, incubation of the substrate at room temperature for 10 min, and a cutoff value for the ELISA OD450⩾0.35, were carried out using a checkerboard titration method. The sensitivity and specificity of this method relative to the Svanova TGEV/PRCV antibody diagnosis kit were 93.5% and 93.8%, respectively.