Evaluation of bovine embryos produced in high performance serum-free media

This review evaluates the quality of bovine embryos developed from in vitro-matured (IVM) and -fertilized (IVF) oocytes cultured in either serum-free or serum-containing media. Bovine embryos cultured in serum-supplemented medium contain numerous cytoplasmic lipid droplets and immature mitochondria compared to those cultured in serum-free medium. The accumulation of cytoplasmic lipids in embryos developed in serum-containing medium may be a result of incorporation of lipoproteins from the serum and may result in impaired function of mitochondria. The improved serum-free media (IVMD101 and IVD101) offer several advantages over culture in serum-containing medium, including increased rates of blastocyst formation and higher cell numbers. Additionally, the survival and hatching rates of embryos produced in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containing medium, suggesting that the abnormal accumulation of cytoplasmic lipids in embryos may have a negative effect on the sensitivity of embryos to chilling and freezing. These serum-free culture systems have proven to be beneficial for the production of good quality embryos from IVM-IVF bovine oocytes. Furthermore, recent studies have shown a correlation between mitochondrial function (oxygen consumption) and embryo quality. A new method using scanning electrochemical microscopy may be capable of assessing the viability and developmental potential of bovine embryos.     

Effects of a macrolide antibiotic on enamel formation in rat incisors--primary lesion of ameloblast at the transition stage

A novel macrolide antibiotic was administered orally to 5-week-old Jcl:Wistar rats at a dose of 5,000 mg/kg/day for 5 weeks, and then a half of animals were maintained without any treatment for 10 weeks. A white discolored lesion with horizontal stripes developed on the surface of the upper and lower incisors after dosing for 4 weeks, and these macroscopical incisal lesions disappeared with the eruption in 4 weeks after stop of administration. Histopathologically, increase in number of karyopycnosis of ameloblast at the transitional stage, vacuolar degeneration of ameloblast and cystic change in the maturation stage, and impaired iron pigment secretion at the pigmentation stage were observed. Microradiography, calcio-traumatic zones, which means hypocalcification, were observed on the superficial layer of enamel. These results suggest that the primary lesion induced by a novel macrolide antibiotic is the increased karyopycnosis of ameloblast at the transitional stage, and followed by later stage.     

Generation of canine dendritic cells from peripheral blood mononuclear cells

Dendritic cells (DCs) are the most potent antigen-presenting cells that are expected to be therapeutic agents for tumor immunotherapy. In this study, we generated DCs of sufficient number for DC-based immunotherapy from peripheral blood mononuclear cells (PBMC) in dogs. PBMC were cultured in the presence of phytohemagglutinin (PHA). On day 6, large adherent cells with dendrite-like projections were seen, and the number of these large cells with projections increased on day 8. These cells were positive for esterase staining. They expressed MHC class II, CD11b, CD8 and weakly CD4 on their surface. They tended to make contact with lymphocytes under culture conditions. We obtained about 2-5 x 10(6) of DCs from 10 ml of peripheral blood. These DCs phagocytosed HEK-293 cells by overnight co-culturing. These cells generated from PBMC are possible canine DCs and are applicable to clinical trials of DC-based whole tumor cell immunotherapy in dogs.     

Effects of a high-protein diet versus dietary supplementation with ammonium chloride on struvite crystal formation in urine of clinically normal cats

OBJECTIVE: To evaluate the effects of a high-protein diet versus dietary supplementation with ammonium chloride (NH4Cl) on struvite crystal formation in the urine of clinically normal cats by measuring the urine concentration of hydrochloric acid (HCl)-insoluble sediment, urine pH, struvite activity product (SAP), number of struvite crystals in urine, and urine volume. ANIMALS: 23 healthy adult cats. PROCEDURE: Urine was fractionated by centrifugation with subsequent extraction of the sediment with 1 N HCl (study 1). Diets containing either 29% crude protein or 55% crude protein were fed to cats in a crossover trial of 3 weeks/period (study 2). Diets supplemented with either sodium chloride (NaCl) or NH4Cl were fed, by use of a 3 x 3 Latin-square design with 3 wk/period (study 3). In studies 2 and 3, urine samples were collected for the last 7 days of each period. RESULTS: The HCl-insoluble sediment contained Tamm-Horsfall glycoprotein (THP; study 1). The high-protein diet (study 2) and dietary supplementation with NH4Cl (study 3) resulted in a decrease in urine pH, SAP, and the number of struvite crystals in urine. However, the high-protein diet decreased urine concentrations of HCl-insoluble sediment containing THP (study 2), in contrast to the NH4Cl supplementation that increased urine volume without a significant effect on the urine concentration of the HCl-insoluble sediment (study 3). CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that compared with dietary supplementation with NH4Cl, the high-protein diet is preferable as a urine acidifier for the prevention of struvite crystal formation in clinically normal cats.     

Hyaluronidase is not essential for the lethality of Erysipelothrix rhusiopathiae infection in mice

To investigate the role of hyaluronidase in the pathogenicity of Erysipelothrix rhusiopathiae, transposon Tn916 was transferred from Enterococcus faecalis CG110 to a virulent strain of E. rhusiopathiae, and hyaluronidase-deficient mutants were isolated. A virulence assay in the mice showed that of the seven hyaluronidase-deficient mutants tested, six mutants were avirulent, but that one mutant, designated AST121, was as virulent as its parental strain. Western immunoblotting with a monoclonal antibody specific to the capsule, a major virulence factor of the organism, revealed that all of the avirulent mutants had lost the capsular antigen, whereas the mutant AST121 did not. These results suggest that the lack of virulence of the six hyaluronidase-negative mutants could be due to a loss of the capsule and that hyaluronidase does not contribute to the lethality of E. rhusiopathiae infection in mice.     

Successful treatment of two dogs with allergic dermatitis by anti-allergic peptides (MS-antigen)

The effects of non-specific immunotherapy with anti-allergic peptides extracted from the urine of human allergic patients (MS-antigen), in two dogs with allergic dermatitis (AD) have been described. Clinically, severe pruritus accompanied by secondary bacterial pyoderma did not respond to conventional therapy with systemic antibiotics. The first clinical change appeared as a significant reduction in pruritus within 3 months, around the time of the 15th injection in both cases. The clinical condition was stabilized after 5 months, allowing the gradual withdrawal of concurrent therapies and an increase of injection intervals. The correlation between the results of intradermal skin tests before and after treatment and the improvement of clinical signs was not obvious.     

Evaluation of isotonic sodium bicarbonate solution for alkalizing effects in conscious calves

After intravenous (i.v.) infusion of various volumes of 1.35%-isotonic sodium bicarbonate solution (ISB), acid-base equilibrium, blood pressure, plasma volume and biochemical parameters in healthy Holstein calves were studied. Four calves each were randomly assigned to the low-dose (LD; i.v. infusion of 5 ml/kg ISB), middle-dose (MD; i.v. infusion of 10 ml/kg ISB) and the high-dose groups (HD; i.v. infusion of 15 ml/kg ISB). Administration volumes of ISB in the LD, MD and HD groups were decided based on the first half volumes of 5, 10 and 15 mEq of base requirement by the acceptable equation. Systemic, pulmonary artery and central venous pressures, cardiac output and plasma osmotic pressure were not changed by ISB infusion and remained constant throughout the experiment for all groups. There was good correlation (r(2) = 0.950) between relative changes in base excess and infused volume of bicarbonate (y=2.491x). The coefficient of distribution for bicarbonate ions was calculated to be 0.401 (=1/2.491). Therefore, it is suggested that a value of 0.4 would be most appropriate when calculating the base requirements in calves. Therefore, the first half volumes of ISB correcting base deficits of 5, 10 and 15 mEq in calves were estimated to be 6, 12 and 18 ml/kg, respectively. On the basis of the findings in this study, ISB may be used to correct metabolic acidosis without altering the plasma osmotic pressure, hemodynamic status and respiratory function in the calves.