Influence of cysteine in conjunction with growth factors on the development of in vitro-produced bovine embryos.

Cysteine supplementation to in vitro maturation (IVM) media of bovine oocytes increases cellular glutathione production. Beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mm cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/ml); EGF (10 ng/ml); and IGF-I + EGF (100 + 10 ng/ml). Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the maturation media (12 h C IGF-I + EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I + EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD) and manganese (Mn) SOD in embryos was assessed. No treatment effect was observed on the expression of these genes. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS.

Effect of droplet vitrification on development competence, actin cytoskeletal integrity and gene expression in in vitro cultured mouse embryos.

The effect of modified droplet vitrification was assessed on cellular actin filament organization, apoptosis related gene expression and development competence in mouse embryos cultured in vitro. Mouse zygotes, 2-cell embryos and morulae were vitrified in ethylene glycol (VS-1) and ethylene glycol plus DMSO (VS-2) and thawed by directly placing the vitrified drop into 0.3M sucrose solution at 37 degrees C. High recovery (93-99%) of morphologically normal embryos was evident following vitrification and thawing. No detectable actin filament disruption was observed in the embryos at any development stage following vitrification and thawing and/or in vitro culture. The expression pattern of Bax, Bcl2 and p53 genes was altered (P<0.05) in vitrified zygotes and 2-cell embryos, but not in morulae. Although a large proportion of the vitrified zygotes (59.5+/-4.4% in VS-1 and 57.9+/-4.5% in VS-2; mean+/-S.E.M.) and 2-cell embryos (63.1+/-4.4% in VS-1 and 59.2+/-4.3% in VS-2) developed into blastocysts, development of control embryos (70.2+/-5.0% of zygotes and 75.5+/-4.4% of 2-cell embryos) into blastocysts was higher (P<0.05). In contrast, development of the control and vitrified morulae into blastocysts (more than 85%) was similar. We concluded that the modified droplet vitrification procedure supported better survival of morula stage compared to zygotes and 2-cell mouse embryos.

Short communication: analysis of immune function in lactating dairy cows fed diets varying in phosphorus content.

The aim of this study was to evaluate the effect of varying dietary P on bovine immune function. Nine first- or second-lactation Holstein cows were fed diets varying in P in a 3 x 3 Latin square design. Diets were formulated to contain either low (0.34%, no supplementary P), medium (0.43%), or high (0.52%) P. All 3 diets were formulated to meet or exceed current NRC requirements for P content. Between d 21 and 26 of each period, blood samples were collected and serum inorganic P concentration, lymphocyte proliferation, and neutrophil bactericidal activity were measured. Serum P increased with increasing dietary P intake and was greatest in the first lactation compared with subsequent lactations. There was a stage of lactation-dependent increase in lymphocyte proliferation after stimulation with concanavalin A, phytohemagglutinin, or pokeweed mitogen. However, dietary P did not alter lymphocyte proliferation or neutrophil bactericidal activity in vitro. In conclusion, decreasing dietary P to reduce manure P content and the risk of P losses from farms to surface water does not have an adverse effect on the innate or cell-mediated immune responses of lactating dairy cattle.

Gene expression and development of mouse zygotes following droplet vitrification.

The concept of ultra-rapid vitrification has emerged in recent years; the accelerated cooling rate reduced injury attributed to cryopreservation and improved post-freezing developmental competence of vitrified oocytes and embryos. The objectives of the present study were to develop a simple and effective ultra-rapid vitrification method (droplet vitrification) and evaluate its effects on post-thaw development and apoptosis-related gene expression in mouse zygotes. Presumptive zygotes were equilibrated for 3 min in equilibration medium and washed 3 times in vitrification solution. A drop (5 microL) of vitrification solution containing 10-12 embryos was placed directly onto surface of liquid nitrogen, with additional liquid nitrogen poured over the drop. For thawing and cryoprotectant removal, vitrified drops were put into dilution medium for 3 min, followed by M2 medium for 5 min. Although cleavage rate did not differ significantly among the control (90.8+/-2.8%; mean+/-S.E.M.), toxicity control (83.5+/-3.2%), and vitrified (86.2+/-3.1%) zygotes, rates of blastocyst and hatched blastocyst formation were lower (P<0.01) in vitrified zygotes (49.7+/-4.7% and 36.0+/-4.7%) and toxicity controls (47.3+/-4.6% and 40.3+/-4.6%) compared with controls (65.5+/-4.1% and 54.2+/-4.3%). Exposure of zygotes to vitrification solution, as well as the vitrification process, down-regulated the expression of Bax, Bcl2, and p53 genes in blastocysts. Although droplet vitrification was efficient and easy, it altered the transcriptional activities of Bax, Bcl2, and p53 genes in vitrified embryos, indicating a strong relationship between reduced developmental competence and the altered transcriptional activities of these genes.

In situ assays demonstrate that interferon-gamma suppresses infection-stimulated hepatic fibrin deposition by promoting fibrinolysis.

BACKGROUND: Inflammatory cytokines potently impact hemostatic pathways during infection, but the tissue-specific regulation of coagulation and fibrinolysis complicates studies of the underlying mechanisms. METHODS AND RESULTS: Here, we describe assays that quantitatively measuring prothrombinase (PTase), protein C-ase (PCase) and plasminogen activator (PA) activities in situ, thereby facilitating studies of tissue-specific hemostasis. Using these assays, we investigate the mechanisms regulating hepatic fibrin deposition during murine toxoplasmosis and the means by which interferon-gamma (IFN-gamma) suppresses infection-stimulated fibrin deposition. We demonstrate that Toxoplasma infection upregulates hepatic PTase, PCase, and PA activity. Wild type and gene-targeted IFN-gamma-deficient mice exhibit similar levels of infection-stimulated PTase activity. By contrast, IFN-gamma-deficiency is associated with increased PCase activity and reduced PA activity during infection. Parallel analyses of hepatic gene expression reveal that IFN-gamma-deficiency is associated with increased expression of thrombomodulin (TM), a key component of the PCase, increased expression of thrombin-activatable fibrinolysis inhibitor (TAFI), a PC substrate, and reduced expression of urokinase PA (u-PA). CONCLUSIONS: These findings suggest that IFN-gamma suppresses infection-stimulated hepatic fibrin deposition by suppressing TM-mediated activation of TAFI, thereby destabilizing fibrin deposits, and concomitantly increasing hepatic u-PA activity, thereby promoting fibrinolysis. We anticipate that further application of these in situ assays will improve our understanding of tissue-specific hemostasis, its regulation by cytokines, and its dysregulation during coagulopathy.

Increased 15-HPETE production decreases prostacyclin synthase activity during oxidant stress in aortic endothelial cells.

Selenium (Se) is an integral component of glutathione peroxidase and is able to detoxify peroxides that can affect arachidonic acid (AA) metabolism, thereby influencing eicosanoid biosynthesis. This study investigated the effects of oxidant stress, a consequence of Se deficiency, on eicosanoid formation and important key enzyme expression in bovine aortic endothelial cells (BAEC). Bovine aortic endothelial cells cultured in Se-deficient media and stimulated with tumor necrosis factor alpha or H2O2 produced significantly less prostacyclin (PGI(2)) and more 15-hydroxyeicosatetraenoic acid, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and thromboxane than Se-supplemented BAEC. Additionally, reverse transcription polymerase chain reaction and immunoblotting determined that the mRNA and protein levels of the eicosanoid forming enzymes cyclooxygenase-1 (COX1), cyclooxygenase-2 (COX2), and PGI synthase were not significantly changed. The addition of 15-HPETE to Se-supplemented BAEC inhibited the production of PGI(2) suggesting that the accumulation of lipid hydroperoxides during Se-deficiency may be the underlying factor in the altered eicosanoid production during Se deficiency. Furthermore, inhibition of COX and addition of PGH(2) to Se-deficient or Se-supplemented BAEC still resulted in lower PGI(2) formation by Se-deficient cells. Together, these results suggest that Se deficiency modifies eicosanoid production by affecting the activity of key enzymes, particularly PGI synthase, rather than their transcription or translation.

Staphylococcus aureus agr genotypes with enterotoxin production capabilities can resist neutrophil bactericidal activity.

Staphylococcus aureus pathogenicity is mainly due to the production of a number of secreted and cell surface-associated proteins under the regulation of the agr gene. A region of the agr gene was used to subgroup S. aureus strains according to restriction fragment length polymorphisms. Additionally, strains were subtyped according to the coagulase gene in order to strengthen discriminatory power. Virulence capabilities of agr genotype subgroups were evaluated using an in vitro neutrophil bactericidal assay, which showed that prevalent genotypes were significantly better at evading this primary host defense. Multiplex PCR was then used to detect enterotoxin genes among the genotype subgroups in order to determine possible virulence candidates that enable strains to combat neutrophil killing. The prevalent genotype strains were found to possess higher production capabilities for enterotoxin A than did low-prevalence strains. The significance of enterotoxin A production capabilities in affecting pathogenicity of S. aureus strains was evaluated and found to have a profound effect on neutrophil killing abilities. The use of a large epidemiological database as a tool for subgrouping strains with varying degrees of pathogenicity has allowed the identification of relevant and previously undefined virulence factors that affect a pathogen's capability to overcome host immune defenses.