鸡β-防御素在大肠杆菌中的高效表达及复性

鸡β-防御素(gallinacin-3,Gal3)是鸡体内重要的防御因子,本试验研究了鸡Gal3融合蛋白在大肠杆菌中高效表达的规律,并探索了重组蛋白质的复性工艺.结果表明,加入诱导剂后1h,开始有可检出量的重组蛋白质表达,2h的表达水平已接近最大量;化学诱导剂IPTG 0.2～1.2mol/L对重组蛋白的产量没有明显影响.经Lablmage软件分析,融合蛋白的产量可达菌体总蛋白的35%以上,且主要以包涵体形式存在.将所获包涵体用8mol/L尿素溶解后,分步稀释于含有氧化型谷胱甘肽和还原型谷胱甘肽的复性系统中,重组蛋白得到完全复性.12mg/L复性的重组鸡Gal3能抑制肠炎沙门氏菌的生长,24mg/L能抑制肠致病型大肠杆菌的生长.     

Breed differences of bull frozen‐thawed semen

The objective of this study was to investigate the quality of frozen‐thawed semen from different bull breeds. Commercial frozen‐thawed bull semen samples (26 per breed, 130 totally) of five breeds (Holstein [Η], Brown Swiss [BS], Limousin [L], Belgian Blue [BB], Blonde d' Aquitaine [BA]) were used. After thawing, each semen sample was subjected to thermal resistance test (TR) for 0.5 and 1 hr at 38°C and hypo‐osmotic swelling test (HOST) for 1 hr at 150 mOsm at 37°C. Additionally, all samples were evaluated at times 0 hr (thawing), 0.5 hr (TR), 1 hr (TR) for kinetics by CASA [progressive, immotile, rapid, medium, slow moving spermatozoa, curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), linearity (LIN), straightness (STR), beat cross‐frequency (BCF), amplitude of lateral head displacement (ALH), wobble (WOB)]. Moreover, directly after thawing, all semen samples were evaluated for morphometry, morphology, viability and DNA fragmentation. Statistical analysis was conducted using a mixed model for repeated measures. The results showed (a) higher VCL after thawing in H, L breeds compared to BB and BA, (b) higher VAP after thawing in L compared to BB, BA, (c) higher values of progressive spermatozoa after TR in H, BS compared to BB, BA, (d) higher values of rapid spermatozoa after thawing and 0.5 hr of TR in H, BS, L compared to BB, BA, (e) lower viability in BA after thawing compared to H, BS, BB, (f) lower morphological abnormalities in H compared to L, BB, (g) higher head length in Η compared to BB. No significant differences were observed in the results from HOST and DNA fragmentation between breeds. In conclusion, quality characteristics of frozen‐thawed bull semen are dependent on the breed. Frozen semen from BB and BA breeds should be handled more carefully after thawing, as it is more sensitive to stress.     

Appraisal and standardization of curvilinear velocity (VCL) cut‐off values for CASA analysis of Japanese quail (Coturnix japonica) sperm

One of the basic steps in objective analysis of sperm motility is the subdivision of a motile sperm population into slow, medium and rapid categories based on their velocity. However, for CASA analysis of quail sperm, the velocity values for categorization of slow, medium and rapid sperm have not yet been standardized. To identify the cut‐off values of “velocity curvilinear” (VCL) for quail sperm categorization, we captured and analysed 22,300 tracks of quail sperm using SCA^®‐CASA. The median and mean VCL values were 85 and 97 μm/s. To define the VCL cut‐off values, we used two methods. In the first, we identified the upper (rapid sperm) and lower (slow sperm) cut‐off values using: (i) median VCL ± 25% or ± 50% or ± 75% of median VCL value; (ii) first and third quartile values of VCL data (i.e. 25% cut‐off setting); and (iii) 33% and 66% of VCL data. Among these settings, sperm categories and their corresponding motility characteristics recorded using the “25%” setting (i.e. slow ≤36 ≤ medium ≤154 ≤ rapid) were found the most realistic and coherent with male ranking by fertility. In the second method, we calculated heteroscedasticity in the total VCL data using PCA and the two‐step clustering method. With this approach, the mean of the high and low clusters was 165 and 51 μm/s, respectively. Together, the mean from two methods suggested that, for SCA^®‐CASA categorization of quail sperm, sperm should be classed as “rapid” at VCL ≥160 μm/s and “slow” at VCL ≤45 μm/s.     

Pronuclear Embryo Yield in Canindé and Saanen Goats for DNA Microinjection

The objective of this study was to examine the effect of donor breed on pronuclear‐stage embryo yield to be used for DNA microinjection in a transgenesis goat program. Twelve Canindé and twelve Saanen goats were heat synchronized using a progestagen‐cloprostenol treatment. Forty‐eight hours before the sponge removal, superovulation was induced with a total administration of 4.4 mg/kg bodyweight NIH‐FSH‐P1, given twice daily in decreasing doses over 3 days. In addition, goats received 100 μg of GnRH and they were hand‐mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after sponge removal. Embryos were microinjected with a DNA construct and noticeable swelling of the nuclei was the criterion for successful microinjection. The total diameter, cytoplasm diameter, zona pellucida thickness and pronuclei diameter were measured for each microinjected embryo. A higher (p < 0.05) percentage of fertilized ova was observed in Canindé (89.9%) than Saanen (36.2%) goats. In addition, Canindé donors produced a higher percentage of pronuclear embryos when compared with Saanen: 72.5% vs 20.6% (p < 0.05), respectively. Successful microinjection was verified in 96.7% and 73.3% of times in Canindé and Saanen embryos, respectively (p < 0.05). Significant differences were observed for all morphometric parameters except for cytoplasm diameter. In conclusion, under our study experimental conditions, Canindé were more efficient than Saanen goats concerning the pronuclear embryo yield and manipulation. The use of Canindé goats in transgenesis programs could be increase the interest in their breeding and could be contribute to saving them from extinction.     

Attitudes and perceptions of veterinary paraprofessionals in New Zealand to postoperative pain in dogs and cats

AIM: To survey the attitudes and perceptions of veterinary paraprofessionals in New Zealand to postoperative pain in dogs and cats.

METHODS: In December 2011, veterinary paraprofessionals (VP) from throughout New Zealand were invited to participate in an online survey. Eleven questions, which were divided into five sections, were used to determine demographic information, the respondents’ assessment of pain after commonly performed surgeries in dogs and cats, their opinions on provision of analgesia, who had responsibility for pain monitoring and the use of any formal pain scoring system in the practice.

RESULTS: Data from 165 respondents were able to be used, and 162 (98%) respondents to the survey were female. According to the respondents’ estimates, fracture repair in dogs and repair of diaphragmatic hernias in cats had the highest pain score following surgery. Neutering procedures involving dogs were scored higher than for cats (p<0.01). All respondents agreed that animals benefit from perioperative analgesia. The veterinary nurse was reported to be predominantly responsible for monitoring pain in animals postoperatively by 116/165 (70.3%) respondents. Of 165 respondents, 154 (93%) considered that their knowledge of pain and assessment of pain could be enhanced.

CONCLUSIONS: This survey reflects the attitudes and perceptions of a sample of VP in New Zealand to postoperative pain in dogs and cats. The results indicate that all respondents believe that surgery results in sufficient pain to warrant analgesic therapy. Routine neutering surgeries were considered to be more painful in dogs than in cats. The current survey also provides information to educators on potential areas of focus, given that 93% of respondents felt that their knowledge of pain and assessment of pain could be enhanced.     

SAGP基因克隆及反义表达载体的构建

为了研究不同启动子驱动的反义SAGP抑制内源ADP-Glc PPase小亚基编码基因表达的效果,创造适于外源基因表达的突变体,利用RT-PCR方法,由马铃薯块茎cDNA克隆获得ADP-Glc PPase小亚基编码基因(SAGP)的1 821 bp cDNA。核酸数据库检索和序列比对分析表明,已克隆的SAGP基因cDNA推测编码607个氨基酸组成的多肽,其中第312和411的天冬酰氨均突变为丝氨酸。以pCambia为基础,分别构建了CaMV35S和GBSSI块茎启动子驱动的小亚基cDNA反义结构植物表达载体。     

Pathogenesis of reproductive failure induced by Trypanosoma vivax in experimentally infected pregnant ewes

The present study was aimed at investigating the effect of experimental infection by Trypanosoma vivax in different stages of pregnancy, determining the pathogenesis of reproductive failure, and confirming transplacental transmission. We used 12 pregnant ewes distributed into four experimental groups: G1, was formed by three ewes infected with T. vivax in the first third of pregnancy (30 days); G2 comprised three infected ewes in the final third of pregnancy (100 days); G3 and G4 were composed of three non-infected ewes with the same gestational period, respectively. Each ewe of G1 and G2 was inoculated with 1.25 × 10⁵ tripomastigotes. Clinical examination, determination of parasitemia, serum biochemistry (albumin, total protein, glucose, cholesterol, and urea), packed cell volume (PCV), serum progesterone, and pathological examination were performed. Placenta, amniotic fluid, blood and tissues from the fetuses and stillbirths were submitted to PCR. Two ewes of G1 (Ewe 1 and 3) presented severe infection and died in the 34^th and 35^th days post-infection (dpi), respectively; but both fetuses were recovered during necropsy. In G2, Ewe 5 aborted two fetuses on the 130^th day (30 dpi) of pregnancy; and Ewe 6 aborted one fetus in the 140^th day (40 dpi) of gestation. Ewes 2 and 4 delivered two weak lambs that died five days after birth. Factors possibly involved with the reproductive failure included high parasitemia, fever, low PCV, body score, serum glucose, total protein, cholesterol, and progesterone. Hepatitis, pericarditis, and encephalitis were observed in the aborted fetuses. The presence of T. vivax DNA in the placenta, amniotic fluid, blood, and tissues from the fetuses confirms the transplacental transmission of the parasite. Histological lesion in the fetuses and placenta also suggest the involvement of the parasite in the etiopathogenesis of reproductive failure in ewes.     

Biomimetic Pathways for Assembling Inorganic Thin Films

Living organisms construct various forms of laminated nanocomposites through directed nucleation and growth of inorganics at self-assembled organic templates at temperatures below 100°C and in aqueous solutions. Recent research has focused on the use of functionalized organic surfaces to form continuous thin films of single-phase ceramics. Continuous thin films of mesostructured silicates have also been formed on hydrophobic and hydrophilic surfaces through a two-step mechanism. First, under acidic conditions, surfactant micellar structures are self-assembled at the solid/liquid interface, and second, inorganic precursors condense to form an inorganic-organic nanocomposite. Epitaxial coordination of adsorbed surfactant tubules is observed on mica and graphite substrates, whereas a random arrangement is observed on amorphous silica. The ability to process ceramic-organic nanocomposite films by these methods provides new technological opportunities.