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.

The potential relationship between the establishment of Foot-and-mouth disease virus (FMDV) persistent infection and gene variation was identified by investigating the variation of VP1 and 3ABC genes from yellow cattle persistent infection isolates. Five yellow cattle were inoculated on their tongue with 1.0×104 ID50/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. The VP1 and 3ABC genes of the 12 isolates were then amplified by reverse-transcriptase polymerase chain reaction (RT-PCR). Cloning and sequencing revealed that the homology of the VP1 nucleotide 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 the VP1 nucleotide sequence of the isolates was only 85%, and that of the deduced amino-acid sequence only 90%.There were several nucleotide mutations in the VP1 gene 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. The 3ABC gene had only 13 nucleotide transversions and five amino-acid mutations. It was presumed that persistent FMDV infection might have little connection with variation in the VP1 and 3ABC genes, and was probably related to other structural protein gene and key factors.

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).

Domestic yak (Bos grunniens) metallothionein-I (MT-I) and metallothionein-III (MT-III) cDNA 3′-end full-length sequences (331 and 378 bp; GenBank accession MT-I no. AY758557, MT-III no. DQ492300) from the total RNA of liver and brain tissues were amplified and cloned by reverse transcription polymerase chain reaction (RT-PCR) and 3′-rapid amplification of cDNA ends (RACE) using the primers YMT-I SP and M13 primers M4, and YMT-III SP and M13 primers M4, respectively, which included the MT-I (183 bp) coding sequence, MT-III (207 bp) cDNA coding sequences, and also the tailing signal AATAAA and Poly(A) at the 3′-ends of the MT-I and MT-III, respectively. The analyses showed that the yak cDNA sequence coding for MT-I protein was composed of 61 amino acids, including 20 cysteines having a conserved tripeptide structure, for example C–X–C, C–C–X–C–C, C–X–X–C and so on. The yak cDNA sequence coding for MT-III protein was composed of 68 amino acids, including 19 cysteines having both MT-III-specific conserved tripeptides and the same conserved tripeptides as MT-I, including T, CPCP, GEGAEA and so on. They were all comparatively conservative in molecular evolution. These structures indicated that MT-III had the same physiological functions regarding heavy metals (detoxification and so on) as MT-I, and also different specific functions from MT-I, including inhibition of neuron growth.

Single-nucleotide polymorphisms (SNPs) of exon 8 of the GHR gene were detected in Chinese Holstein cows by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). The results showed that exon 8 of the GHR gene digested by TasI could be divided into two kinds of alleles and three kinds of genotypes. The frequencies of allele A and T were 0.6339 and 0.3661, respectively. The frequencies of genotypes AA, AT and TT were 0.459, 0.350 and 0.191, respectively. Sequencing showed one single nucleotide mutation T→A at 4962 bp of the gene in genotype TT when compared with genotype AA, and this mutation resulted in an amino acid change of phenylalanine (TTT)→tyrosine (TAT). The result of χ2 testing indicated that the genotypic frequency of the GHR gene digested by TasI did not fit with Hardy–Weinberg equilibrium in this population (P<0.05). The least-square mean of the fat percentage of this population was higher for genotype TT than for genotype AA (P<0.05).

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.

In exploring an effective and reliable karyotyping method in Brassica crop plants, Brassica oleracea was successfully karyotyped using genomic in situ hybridization (GISH). B. oleracea genomic DNA was labelled as probe using DIG-high prime mix kit, with B. rapa genomic DNA acting as blocking agent. Specific fluorescent signals were detected on each pair of homologous chromosomes, and nine pairs of chromosomes of B. oleracea were clearly identified according to the signal characteristics. A practical and accurate method for conducting karyotyping of small chromosomes has been demonstrated.

The Dorset ram of the callipyge phenotype presents with muscular hypertrophy in the buttocks, and its inheritance is polar overdominant. A partial DNA fragment of 250 bp was obtained from the goat (Capra hircas) callipyge gene (CLPG; GenBank accession no. EU753362), which shared 96.04% and 88.65% identity with the corresponding regions of ovine (Ovis aries) and porcine CLPG, respectively. A polymorphism in the DNA fragment was detected by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Sequencing results indicated no A→C mutation corresponding to the ovine CLPG gene, although one A→C transversion was located 147 bp downstream from the CLPG site. The polymorphism, named SNP216 after its position (where SNP indicates single-nucleotide polymorphism), was investigated in Boer (n=63), Laiwu Black (n=70), Lubei White×Boer Hybrid (n=40), Lubei White (n=29) and Inner Mongolia Alashan White cashmere (n=115) goat populations. The results indicated that allele A was dominant in four of the goat populations, the Inner Mongolia Alashan White cashmere goats being the exception. The first four populations were in a state of Hardy–Weinberg equilibrium (P>0.05). In Inner Mongolia Alashan White cashmere goats, least-square means of birth weight, production of cashmere and body weight gain from birth to weaning did not differ significantly between the AA and AC phenotypes (P>0.5).

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.

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.

To verify its antibacterial activity, recombinant human lactoferrin (rhLF) was extracted from the milk of transgenic mice (Mus musculus) (PCL25 and 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 on Escherichia coli and Salmonella.

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.

The F1 hybrid offspring of the distant hybridization between Cichlasoma trimaculatum (male) and Heros managuense (female) were successfully obtained through artificial fertilization, and the chromosome specimen of kidney cells of the two parents and their offspring were prepared using a low colchicine concentration air drying method, Howell and Black's (1980) silver–nucleolar organizer region (Ag–NOR) staining method and Sumner's (1972) C-band staining method. The karyotypes, Ag–NORs and C-bands of C. trimaculatum, H. managuense and their offspring were observed. The results showed that the diploid numbers of the two parents and the F1 hybrid offspring were all 2n=48. For C. trimaculatum, the karyotype formula was 2n=2m+8sm+26st+12t, NF=58. Two Ag–NORs were seen on the tips of the short arm of chromosome st7, and the entire chromosomes of st9 and st13 and the centromeres of the other chromosomes had positive C-bands. For H. managuense, the karyotype formula was 2n=2m+6sm+28st+12t, NF=56. Two Ag–NORs were located on the tips of the short arm of chromosome sm2 and positive C-bands were found in the centromere of all chromosomes. In the F1 hybrid offspring, the chromosome formula was 2n=2m+8sm+30st+8t, NF=58. Two Ag–NORs were seen on the tips of the short arm of chromosome sm2; all chromosomes presented positive C-bands in the centromere. No heteromorphic sex chromosome was found. After comparison with the two parents, the F1 hybrid offspring was suggested to be the filial generation of the two hybrid parents rather than the gynogenesis of H. managuense induced by heterogeneous sperm of C. trimaculatum.

Primers bearing restriction enzyme sites for EcoR I and Hind 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 the gcgasa gene. Two fragments with lengths 319 and 238 bp were obtained and were further cloned into plasmid pET-32(a). Following transformation into Escherichia coli BL21(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.

Using semi-quantitative reverse transcriptase polymerase chain reaction (semi-qRT-PCR), cell transfection, Hoechst 33342, acridine orange (AO) and flow cytometry methods, apoptotic changes in porcine pre-adipocytes treated with 9-cis retinoic acid (9-cisRA)–retinoid X receptor α (RXRα) ligand, transfected with pEnhanced green fluorescent proteins C2–RXRα (pEGFPC2–RXRα) or RXRα–small interfering RNA (RXRα–siRNA), respectively, were investigated. Observations of morphologic changes showed changes in cell structure, cellular shrinkage and condensation of chromatin, pyknosis of the nucleus into a compact body, complete fragmentation within the nuclear membrane, continuous budding and shedding, and, finally conversion of the cell into several apoptotic bodies wrapped in membranes of different sizes. Compared with the control group, the apoptotic rate was significantly lowered in the 10 nmol/l 9-cisRA-treated group, and significantly higher in the 10 μmol/l 9-cisRA-treated group (both P<0.05). Gene expression of RXRα was significantly up-regulated and down-regulated when cultured pre-adipocytes were transfected with pEGFPC2–RXRα or RXRα–siRNA (P<0.05), respectively; the apoptotic rate decreased in the pEGFPC2–RXRα group and significantly increased in the RXRα–siRNA group when compared with a control group (P<0.05). This implies that RXRα inhibits the apoptosis of porcine pre-adipocytes.