牛精子和卵母细胞低温保存研究进展

牛的精子和卵母细胞的低温保存对于畜牧业的发展有重要价值。该文综述了近年来牛精子和卵母细胞低温保存的研究进展,包括保存方法、与低温保存有关的细胞特性、存在的问题和改进的方法等内容。     

Isolation of polymorphic AGC repeats located 3' to bovine SINEs

A bovine genomic library was screened for the presence of (AGC)n repeats. All isolated AGC repeats were located adjacent to the 3′ end of bovine short interspersed nuclear elements (SINE). Polymerase chain reactions (PCR) using either two unique primers or one unique and one SINE primer produced high-resolution products without the secondary artifact ladders typical of dinucleotide microsatellites. Four AGC microsatellites were found to be polymorphic with 2–4 alleles each and polymorphism information context (PIC) values ranging between 0.26 and 0.49. One microsatellite, AR025, was mapped to chromosome 26 with the CSIRO reference families. Because of their strong association with AGC repeats and high frequency in the genome, SINE-3′ PCR may prove to be a novel source of polymorphic trinucleotide markers in the bovine genome.     

Trace 5-Methylaminomethyl-2-selenouridine in bovine tRNA and the selenouridine synthase activity in bovine liver

We measured the amount of Se in bovine liver tRNA. tRNA was chromatographed on a BD-cellulose column and Se-rich tRNA was eluted from the column in front of a main tRNA peak. There was 0.3 mmol Se/mol of tRNA and this level is about one tenth that of Escherichia coli tRNA. This suggests the presence of an enzyme that modifies tRNA with Se in bovine liver. We isolated the activity of this enzyme (selenouridine synthase) by chromatography of bovine liver extracts on a DEAE-cellulose column. ATP and selenophosphate synthetase, as well as selenouridine synthase and tRNA, were necessary for the reaction. ⁷⁵Se was used to label the reaction products, which were analyzed by TLC after digestion with ribonuclease T₂. The position of the ⁷⁵ Se-nucleotide on a TLC plate was identical to that of the Se-nucleotide, 5-methylaminomethyl-2-seleno-Up, prepared from ⁷⁵Se-tRNA in E. coli.     

Utilization of discard bovine bone as a support for immobilization of recombinant Rhizopus oryzae lipase expressed in Pichia pastoris

In this study the possibility of using discard bovine bone as support for immobilization of Rhizopus oryzae lipase expressed in Pichia pastoris was analyzed. Discard bovine bone were milled and then subjected to a chemical treatment with acetone in order to remove lipids and blood traces. Two types of supports were evaluated: bovine bone and calcined bovine bone for 2 h at 600°C. Supports were characterized by: ICP, SEM, XRD, FTIR, XPS, and N₂ adsorption isotherms. Calcined bovine bone presented appropriate characteristics for the lipase immobilization due to the removal of collagen: high porosity, large surface area and suitable porous structure. Biocatalysts were prepared with different initial enzyme load. For the equilibrium adsorption studies, the Langmuir isotherm was used to fit the data results. The immobilization occurs in monolayer to a value of 35 UA mg^?1. The activities of biocatalysts were tested in transesterification reaction of olive oil. For the enzyme load used in the test, a final yield percentage of 49.6 was achieved after six methanol additions and 180 min of reaction, similar values were obtained using Relizyme as support. Therefore, the bovine bone discard is an economical and appropriate choice for use support immobilization of enzymes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1246–1253, 2016     

不同pH值与电位耦合对牛血清白蛋白包被酒石酸长春瑞滨纳米粒制备工艺的影响

在反溶剂法制备纳米粒过程中,pH值在一定程度上会对其产生影响。本文通过在不同酸碱环境下运用反溶剂法制备牛血清白蛋白包被酒石酸长春瑞滨纳米粒,进而借助于电位耦合作用来研究纳米粒制备工艺。研究结果表明:当pH=4.5至7.5时,酒石酸长春瑞滨和牛血清白蛋白带有异种电荷,而当pH=2.5,3.5,8.5,9.5时它们均带有同种电荷。当pH=7.5时,牛血清白蛋白带有负电荷即-8.52 mV,酒石酸长春瑞滨带有正电荷即+4.48mV。此时得到牛血清白蛋白包被酒石酸长春瑞滨纳米粒粒径为193.3 nm,Zeta电位为-30.86 mV,而且在该pH下对纳米粒制备工艺进行了优化,最终它的载药量和包封率达到了45.6%和90.6%。     

Evidence for BoLA-linked resistance and susceptibility to subclinical progression of bovine leukaemia virus infection

The role of the bovine major histocompatibility complex in bovine leukaemia virus (BLV) infection and disease progression was investigated in a herd of Shorthorn cattle (n = 117). The frequency of cows that were seropositive to BLV-glycoprotein antigen was 51%. Twenty-three per cent of the seropositive cows were lymphocytotic. At the herd level, relative resistance to BLV-dependent B-cell proliferation and lymphocytosis among seropositive cows was associated with bovine lymphocyte antigen (BoLA)-DA7, whereas susceptibility was associated with BoLA-DA12.3. These associations were also confirmed at the family level, where BoLA phenotypes were used as haplotypic markers. Among the offspring of one BoLA-heterozygous sire (n = 33), resistance segregated with the DA7 haplotype and susceptibility with the DA12.3 haplotype. In this sire group, maternal transmission of the BoLA-w8 allele was associated with increased susceptibility to B-cell proliferation and lymphocytosis in w8/DA12.3 heterozygotes. These data provide the first evidence that subclinical progression of BLV infection is under the control of the BoLA complex, and suggest that the BoLA system can be used to select for resistance to B-cell proliferation and the development of lymphocytosis in BLV-infected herds.     

Regulation of cellular glutathione peroxidase by different forms and concentrations of selenium in primary cultured bovine hepatocytes

The expression and activity of cellular glutathione peroxidase (GPx1) are regulated by selenium (Se). Generally speaking, organic forms of Se have less toxicity and greater bioavailability compared with inorganic forms. In this study, the effects of different forms and concentrations of Se on the regulation of mRNA level and activity of GPx1 in bovine hepatocytes were evaluated, and the optimal doses of different forms of Se that supported the full expression of GPx1 were determined. Primary cultured bovine hepatocyte monolayers derived from neonatal male Holstein calves (aged 1–2 days) were incubated for 24 h with 0 (control), 0.5, 1, 1.5, 2, 3, 4 or 5 μmol/L of Se from dl-selenomethionine (Se-Met), sodium selenite (Na₂SeO₃) or Kappa-selenocarrageenan (Se-Car). Compared with controls, a significantly lower level of release of lactic dehydrogenase (LDH) was observed at 0.5–5 μmol/L of Se-Met, 0.5–1 μmol/L of Na₂SeO₃ and 0.5 μmol/L of Se-Car, but significantly higher LDH release was observed at 2–5 μmol/L of Na₂SeO₃ and 3–5 μmol/L of Se-Car, and the response occurred in a dose-dependent manner. The intracellular content of reduced glutathione in all hepatocytes treated with Se was significantly lower than that of controls. Significant increases in GPx1 mRNA were obtained in all hepatocytes treated with Se, with maximal effects at 3 μmol/L of Se-Met, 1.5 μmol/L of Na₂SeO₃ and 2 μmol/L of Se-Car, respectively. Furthermore, 3 μmol/L of Se from Se-Met resulted in peak levels of GPx1 mRNA. After reaching a maximal level, higher Se supplementation led to a reduction of GPx1 mRNA. The activity of GPx1 showed similar patterns but of lower magnitude. We conclude that (a) the regulation of mRNA level and activity of GPx1 in primary cultured bovine hepatocytes by different forms of Se varies and (b) the optimal doses of Se to support the full expression of GPx1 in bovine hepatocytes when supplied as Se-Met, Na₂SeO₃ and Se-Car are 3, 1.5 and 2 μmol/L, respectively.     

Genetic characterization of the bovine leukaemia inhibitory factor (LIF) gene: isolation and sequencing, chromosome assignment and microsatellite analysis

The bovine leukaemia inhibitory factor was isolated from a phage library and sequences for the gene, in addition to 1213bp of 5' and 432bp of 3' sequences, were obtained and compared with other mammalian leukaemia inhibitory factor genes. Comparisons indicated amino acid homologies ranging from 89·6% to 77·2% with the human and mouse homologues, respectively. Analysis of 500bp of 5' regulatory regions indicated homologies ranging from 83·6% to 74·4% with the corresponding human and sheep sequences, respectively. Additionally, bovine leukaemia inhibitory factor-specific primers were prepared, and a panel of bovine × hamster somatic cell lines were analysed by the polymerase chain reaction (PCR). Data indicated 93% concordance of leukaemia inhibitory factor with aldehyde dehydrogenase 2 located on bovine chromosome 17, and concordance of 81% with myelin basic protein situated on bovine chromosome 24. Southern analysis of selected hybrids confirmed the PCR results, thus conclusively assigning the bovine leukaemia inhibitory factor gene to chromosome 17. Sequence analysis also revealed a microsatellite in intron 2 of the bovine leukaemia inhibitory factor. Analysis of this region by PCR in 22 unrelated Bos taurus and 19 unrelated Bos indicus cattle detected nine different alleles. Polymorphic information content values were 0·53 and 0·80 in B. taurus and B. indicus , respectively. Additionally, the same leukaemia inhibitory factor primers successfully detected allelic variants at this locus in Bos javanicus , Bos guarus and Bison bison but not in Odocoileus virginianus .     

The effects of bovine serum albumin, bovine uterine fluid, and heat-treated bovine serum on in vitro mouse embryo development

Two-cell mouse embryos were cultured in Whitten's medium with one of three supplements: bovine serum albumin (WM + BSA), heat-treated bovine serum (WM + HTBS) or bovine uterine fluid (WM + BUF). Protein concentrations for cultures of WM + BSA were 50.2, 100.5, 251.2, 502.5, and 1005.0 mug/ml and for WM + HTBS were 70.4, 105.1, 269.0, 524.5 and 1193.9 mug/ml. Protein concentrations ranged from 56.9 to 739.1 mug/ml for 22 WM + BUF samples. Embryo development in all media was significantly correlated with the log total protein concentration. When compared to WM + BSA, development was not significantly inhibited or stimulated in any WM + BUF cultures or in WM with 70.4, 524.5 and 1193.9 mug/ml HTBS. Development was enhanced in WM with 105.1 and 269.0 mug/ml HTBS (P<0.05). The results suggest that at the protein concentrations used, culture media supplemented with BUF and BSA support similar mouse embryo development. Culture medium supplemented with HTBS supported embryo development more than medium with BSA. Uterine factors in the bovine capable of enhancing or inhibiting early embryo development were not detected.     

The bovine butyrophilin gene maps to chromsome 23

Chromosomal assignment of the bovine butyrophilin gene ( BTN ) was performed by analysis of DNA from somatic hybrid cell lines using the polymerase chain reaction. The gene was assigned to bovine chromosome 23 using two sets of primers specific for bovine BTN.     

重组牛肠激酶轻链基因在毕赤酵母中的表达与纯化

目的:构建重组牛肠激酶轻链的基因工程菌,并进行表达和纯化,以获得高纯度和高活性的重组牛肠激酶轻链蛋白。方法:以GenBank公共数据库中的牛肠激酶轻链基因序列(AccessionNo.NM174439)设计引物,利用RT-PCR合成牛肠激酶轻链基因片段,并克隆进pPIC9K载体,同时在基因N端插进6个组氨酸标签,转化毕赤酵母GS115,进行筛选和诱导表达。产物经镍离子螯和层析和Q-SepharoseFF柱纯化,并酶切融合蛋白检测其活性。结果:培养液中重组牛肠激酶轻链蛋白表达量为3.0mg/L。对含有肠激酶酶切位点的IL-11/MBP融合蛋白进行酶切,结果表明,酶解率可达到90%以上。结论:表达并获得了高纯度的重组肠激酶轻链蛋白,为大规模生产打下了基础。     

RNA aptamers specific for bovine thrombin

Bovine thrombin is widely used in clinical wound healing after surgery. There is 85% homology between bovine thrombin and human thrombin, so most antibodies against bovine thrombin cross-react with human thrombin. Rare antibodies against bovine thrombin but not cross-reacting with human thrombin have been reported. RNA ligands (aptamers) have been used to bind to target molecules with sometimes higher specificity than antibodies. Here we report the isolation of aptamers specific for bovine thrombin by systematic evolution of ligands by exponential enrichment (SELEX) from an RNA pool containing a 25-nucleotide randomized region. After seven rounds of selection, two aptamers specific for bovine thrombin were identified with a K(d) of 164 and 240 nM, respectively. Significantly, these aptamers do not bind to human thrombin. Secondary structure prediction revealed potential stem-loop structures for these RNAs. Both RNA aptamers inhibit only bovine thrombin-catalyzed fibrin clot formation in vitro. Competition assay results suggested that the RNA aptamers might bind to the electropositive domain of bovine thrombin, that is, heparin-binding site, instead of fibrinogen-recognition exosite. The resulting bovine-specific thrombin inhibitor might be used in some clinical applications when bovine thrombin activity needs to be contained or in research where human and bovine thrombin need to be distinguished.     

聚合牛血红蛋白抗原性的初步研究

对乙醇醛聚合的牛血红蛋白的抗原性进行了研究。将聚合的牛血红蛋白桉兔血量的10％和20％分别输入兔耳缘静脉,间隔7天后重复输液,共输注3次。ELISA检测未显示抗体产生。将聚合的牛血红蛋白、人血红蛋白和兔血红蛋白分别与免疫佐剂混合常规免疫家兔3次,并用上述抗原包被聚乙烯板,用ELISA方法检测抗体滴度,结果显示聚合牛血红蛋白和人血红蛋白加佐剂免疫家兔后均产生抗体,且有交叉反应,表明这两种血红蛋白有同源性,存在相似的抗厚决定簇。兔血红蛋白免疫家兔后无抗体产生,且无交叉反应,表明机体对自体蛋白有天然的耐受。     

Enhanced Expression of Fc Receptors on Neutrophils from Calves with Leukocyte Adhesion Deficiency

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)-binding receptors, luminol-dependent chemiluminescent (LDCL) responses, and the effect of anti-bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1- to 2.5-fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A-binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat-aggregated bovine IgG (Agg-bovine IgG), sheep red blood cells (SRBC) sensitized with anti-SRBC antibody (SRBC-anti-SRBC Ab), or con A had LDCL responses of 36 (P<0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT-reducing value of neutrophils from a calf with BLAD when stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre-incubation with anti-bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 μg/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC-anti-SRBC Ab and NBT-reducing activity stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).     

BoLA antigens are associated with increased frequency of persistent lymphocytosis in bovine leukaemia virus infected cattle and with increased incidence of antibodies to bovine leukaemia virus

The association between bovine major histocompatibility system (BoLA) type and persistent lymphocytosis in cattle with antibodies to bovine leukaemia virus was examined by comparing antigen frequencies in cattle with persistent lymphocytosis to controls matched for age, sex, breed and presence of antibodies to BLV. The cattle came from nine dairy herds in south-east Queensland, Australia; six herds were Australian Illawarra Shorthorn (AIS), two herds were Jersey and one herd was Friesian. Antigen W6 and Eu28R were more common in cattle with persistent lymphocytosis than in controls. Antigen W8 was less common in AIS cattle with persistent lymphocytosis. A study of 24 offspring from one sire, heterozygous for W10 and Eu28R, showed that offspring inheriting Eu28R from the sire were significantly more likely to have antibodies to BLV than offspring inheriting the opposing W10 haplotype.     

Effect of in vitro selenium supplementation on bovine sperm motility

The effects of selenium (Se) on motility of bovine spermatozoa were evaluated in two experiments. In Experiment 1, increasing doses of Se alone or with vitamin E were found to increase motility in Se concentrations up to 1 mug/ml. However, Se alone had a greater effect than did Se with vitamin E. The sperm motility response to Se concentrations from background to 1 mug/ml on motility best fit a quadratic equation with a maximum at about .65 mug Se/ml. In Experiment 2, the effect of Se addition before and after freezing was evaluated. This experiment demonstrated that treatment of semen with Se before freezing significantly increased the post-thaw motility as compared with portions of the same ejaculate which were not treated. Treatment after thawing produced motilities which were intermediate and not significantly different from either pre-freezing supplementation or no supplementation at all. These data indicate that some semen exhibits a greater percentage of motile sperm when Se is added prior to freezing, however, no information is yet available on the fertility of Se-treated semen.     

Chemotactic behavior of Campylobacter fetus subspecies towards cervical mucus,bovine placenta and selected substances and ion

The chemotaxis of C. fetus subsp. venerealis and C. fetus subsp. fetus was determined in the presence of bovine cervical mucus and bovine placental extract. Some reported substances and ion in those materials, such amino acids, ferrous iron, hormones, sugars and organic acids were also investigated. Bovine cervical mucus, bovine placenta extracts and some substances and ion of these materials namely L–fucose, L– aspartate, L–glutamate, L–serine, ferrous iron, fumarate, pyruvate and succinate were chemoattractants. The chemottraction was significantly larger in higher concentrations of the tested substances and ion and significant differences among tested strains were also observed. Meso-erythritol and hormones bovine placental lactogen, 17β-estradiol, and progesterone did not elicit chemotactical response. In conclusion, this chemotactic behavior may guide the C. fetus navigation in the bovine host''s genital tract and be an important cofactor of tissue tropism for this bacterium.     

Specific integrin subunits in bovine oocytes, including novel sequences for alpha 6 and beta 3 subunits

Integrins facilitate attachment of cells to the extra-cellular matrix, often binding the arginine-glycine-aspartic acid tri-peptide motif, thus facilitating cell migration, mediating cell-cell adhesion, linking the extracellular matrix (ECM) with cytoskeletal elements, and acting as signaling molecules. Adhesion activates signaling mechanisms that regulate integrin function, cytoskeletal assembly, cell behavior, and protein synthesis. Immunofluorescence was used to determine the presence of integrin alpha and beta subunits on the surface of bovine oocytes using a panel of monoclonal antibodies (mAbs) specific for alphaL, alphaM, alphaX, alphaV, alpha2, alpha4, alpha6, beta1, beta2, and beta3 antigens, with multiple antibodies for each subunit. Confocal microscopy indicated the presence of alphaV, alpha6, alpha4, alpha2, ss1, and ss3 integrin subunits on the plasma membrane of bovine oocytes. The presence of these subunits was verified by RT-PCR analysis using primers designed based on known gene sequences of bovine integrin subunits, or by using sequence information using bovine expressed sequence tags (EST) compared with known human and murine integrin subunit gene sequence information. Previously unpublished sequence information for bovine alpha6 and beta3 integrins was determined. The presence of these integrin subunits on the bovine oocyte vitelline membrane supports the hypothesis that sperm-oocyte interactions in the bovine are mediated by integrins.