Efficiency of Two Enucleation Methods Connected to Handmade Cloning to Produce Transgenic Porcine Embryos

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41–42 h oocytes maturation, the oocytes were further cultured with or without 0.4 μg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 ± 2% vs 48.1 ± 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.     

Effects of Matrix Filtration of Low-Quality Boar Semen Doses on Sperm Quality

The aim of this work was to develop a method to enhance the sperm parameters of ejaculates with low sperm quality from Piétrain boars. Seminal doses were filtered through columns of DEAE Sephadex (length 2.5 ± 0.5 cm), CM Sephadex (length 5 ± 0.5 cm), glass wool (length 2 ± 0.5 cm) or glass bead (length 10 ± 0.5 cm), with an exit flow rate of 1 ml/40 s in all cases. For each male, 10 ml of the sperm cell-rich fraction diluted at 1 : 6 were filtered. Sperm quality was assessed before and after filtration. Sperm morphology, sperm motility and sperm concentration were determined using the computer program sca ^® 2002 Production, and sperm viability was evaluated by fluorescence multistaining. Osmotic resistance test and hyperosmotic resistance test were used to determine the osmotic resistance of spermatozoa, whereas l -lactate production estimated the metabolic activity. Results showed a decrease of sperm concentration and osmotic resistance of spermatozoa after filtration in the four matrixes. However, an increase in the frequency of viable spermatozoa with intact acrosome after filtration in glass bead columns and an increase of morphologically normal spermatozoa after filtration in Sephadex CM-50, glass wool and glass bead columns were observed. Despite the decrease in the frequency of progressive motile spermatozoa, l -lactate production and mitochondrial sheath integrity maintained constant after filtration. Our findings indicate that column filtration is an effective method to enhance the sperm quality by selecting viable and morphologically normal spermatozoa without altering DNA, plasma membrane, mitochondrial sheath integrity or inducing premature acrosome reaction.     

Variations in the Proportion of Glycolytic/Non-glycolytic Energy Substrates Modulate Sperm Membrane Integrity and Function in Diluted Boar Samples Stored at 15–17°C

In this work the role of energy substrates in the maintenance of boar-sperm survival during storage at 15-17 degrees C was tested. For this purpose, boar spermatozoa were stored at 15-17 degrees C in several defined media with separate combinations of a monosaccharide, glucose and a non-monosaccharide, either citrate or lactate, energy substrates. Our results indicate that the medium containing the highest concentration of glucose together with low lactate levels was the most suitable to maintain sperm quality for 168 h at 15-17 degrees C. This was confirmed after observation of the results of the percentages of viability and altered acrosomes, the osmotic resistance test, the hyperosmotic resistance test and the rhythm of L-lactate production. The survival ability of boar sperm was greater in this experimental medium than in the standard Beltsville Thawing Solution extender, which contains only glucose as an energy substrate, although at a concentration far higher than that of all the tested experimental media. Our results indicate that the exact composition, more than the pure quantity of energy substrates, is a very important modulatory factor which affects survival ability of boar sperm in refrigeration. Thus, the exact combination of several energy substrates would have to be taken into account when optimizing the design of commercial extenders to store boar spermatozoa at 15-17 degrees C.     

Dermal and serological response against Pseudomonas aeruginosa in sheep bred for resistance and susceptibility to fleece-rot

Genetically select lines of Merino sheep have been bred at Trangie (NSW Agriculture and Fisheries) for resistance (R) or susceptibility (S) to fleece-rot and flystrike. It is believed that fleece characters are primarily responsible for the R or S phenotype. When transferred to the wetter coastal environment of Sydney, R and S sheep with no more than 6 weeks wool cover, continued to show significant differences in the incidence and severity of fleece-rot dermatitis. To test the hypothesis that these sheep might also exhibit differences in their local skin reactions and immune responsiveness, 3 intradermal injections of killed Pseudomonas aeruginosa were administered at monthly intervals. After primary intradermal challenge, R sheep had a higher incidence of skin induration and a stronger inflammatory response (increased induration diameter) than S sheep. Compared to S sheep, R sheep also developed higher levels of circulating antibodies against whole cell antigen and both inner and outer membrane proteins of P. aeruginosa. These responses were maintained in R sheep with each consecutive challenge while S sheep showed a decline in their immune responsiveness. Differences in antibody response against outer membrane proteins were also detected when antigenically naive sheep from each genetic line were sensitised by epicutaneous challenge with P. aeruginosa under experimental wetting conditions. Intradermal challenge of these animals 6 months later with outer membrane proteins, revealed a late maximum (72 h) in the development of induration diameters for R sheep while S animals showed maximal induration diameters by 24 h. However, there was no significant difference in induration response between 24 h and 72 h within each group of sheep.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstruction of Current Flow and Imaging of Current-Limiting Defects in Polycrystalline Superconducting Films

Magneto-optical imaging was used to visualize the inhomogeneous penetration of magnetic flux into polycrystalline TlBa2Ca2Cu3Ox films with high critical current densities, to reconstruct the local two-dimensional supercurrent flow patterns and to correlate inhomogeneities in this flow with the local crystallographic misorientation. The films have almost perfect c-axis alignment and considerable local a- and b-axis texture because the grains tend to form colonies with only slightly misaligned a and b axes. Current flows freely over these low-angle grain boundaries but is strongly reduced at intermittent colony boundaries of high misorientation. The local (<10-micrometer scale) critical current density Jc varies widely, being up to 10 times as great as the transport Jc (scale of approximately 1 millimeter), which itself varies by a factor of about 5 in different sections of the film. The combined experiments show that the magnitude of the transport Jc is largely determined by a few high-angle boundaries.     

Systemic antibacterial drug use in dogs in Australia

A survey of veterinarians' use of antibacterial drugs was conducted by distributing a questionnaire to Australian practitioners. Respondents were asked to indicate their general patterns of use of various systemic antibacterial drugs and drug combinations in dogs and their approach to certain specified clinical disorders. Overall, antibacterials of the p-lactam type (penicillins and cephalosporins) were most commonly used. Other antibacterials with substantial use were doxycycline, sulphonamide-trimethoprim, metronidazole, fluoroquinolones and clindamycin. Drug selection for different disorders was generally appropriate when compared with recommendations in recent texts, although inappropriate use was evident in some circumstances.