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.     

Cefquinome Concentrations in Endometrium after Intrauterine Treatment of Cobactan 4.5%® in Mares and Inflammatory Response of the Endometrium to this Treatment

This study was conducted to measure the concentration of cefquinome in the endometrium of mares after intrauterine treatment and to evaluate associated inflammation. Mares (n = 14) were randomly assigned to one of the following groups: (i) control (n = 4) were either not treated (n = 2) or received (n = 2) lactated Ringer's intrauterine for 1 or 3 days; (ii) treated mares (n = 10) received intrauterine cefquinome for 1 or 3 days. After at least 10 days had passed following the last treatment and ovulation, mares were given Prostaglandin F2α (PGF2α) and were randomly assigned to an alternate treatment. Endometrial biopsy samples were taken at 2, 8, 24 and 48 h, or at 4, 12 and 36 h, after the last treatment. Biopsy samples were taken at the same time points from control mares (n = 2) and lactated Ringer-treated mares (n = 2). Cefquinome concentrations were quantified using a high-performance liquid chromatography (HPLC) assay and inflammation was assessed using haematoxylin and eosin (H&E)-stained sections. Concentrations of cefquinome [559 (1 day) and 595 μg/g (3 days) at 2 h, and 403 (1 day) and 370 μg/g (3 days) at 4 h] were similar between treatment groups at 2 and 4 h after treatment (p > 0.05). At 8 h, as well as at 24 and 48 h, concentrations were greater in the 3-day group (17 vs 301 μg/g, 3 vs 80 μg/g and 0.1 vs 0.2 μg/g, respectively) (p < 0.05). No significant differences (p > 0.05) in the inflammatory response at 2–48 h after treatment were found between groups.     

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.     

Race-specific aspects of polygenic resistance of barley to leaf rust,Puccinia hordei

Partial resistance of barley to leaf rust,Puccinia hordei, is characterized by a reduced rate of epidemic development in spite of a susceptible infection type. Barley cultivars vary greatly for partial resistance and its components. In a test for interaction between host cultivars and pathogen isolates most variation was of a horizontal nature. However, in the combination between Julia and the rust isolate 18, a differential interaction (vertical effect) occured; Julia had lost a small part of its partial resistance. The same interaction was found for latent period (LP), the most important component of partial resistance. Julia showed a shortened LP for isolate 18. Genetic analyses revealed, that Julia carries a polygene for longer LP not present in the other cultivars. The effect of this polygene appears to be broken by isolate 18 indicative for a gene-for-isolate relation, and even for a gene-forgene relation.Samenvatting Partiële resistantie van gerst voor dwergroest,Puccinia hordei, is gekenmerkt door een vertraagde epidemie-opbouw ondanks een vatbaar infectietype. Gerstrassen variëren sterk in partiële resistentie en haar componenten. In een toets ter bestudering van de interactie tussen waardplant en pathogeenisolaat bleek de meeste variatie horizontaal van aard te zijn. In de combinatie van Julia met isolaat 18 werd echter een differentiële interactie waargenomen; Julia had een klein deel van zijn partiële resistentie verloren. Dezelfde interactie werd waargenomen voor de belangrijkste component van partiële resistentie, de latentieperiode (LP); Julia had een iets verkorte LP voor isolaat 18. Genetische analyses toonden aan, dat Julia een polygen voor langere LP bevat; dit gen is niet aanwezig in de andere bestudeerde rassen. Er wordt verondersteld dat het effect van dit polygen door isolaat 18 is doorbroken. Dit wijst op een gen-om-isolaat en zelfs op een gen-om-gen-relatie.     

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)     

Partial resistance of barley to leafrust,Puccinia hordei. I. Effect of cultivar and development stage on latent period

Summary The latent period of barley leafrust, Puccinia hordei, was studied in a number of cultivars at different development stages and growing conditions. The relative latent period of the different cultivars was not affected by temperature. Other environmental factors, like daylength and light-intensity did not seem to have any effect either. The development stage of the plant and the genotype on the other hand influenced the latent period greatly. The latent period became longer for all cultivars tested from the seedling stage onward until a maximum was reached with the young flag leaf at heading. For the most susceptible cultivar, L94, the relative latent period increased from the primary leaf in the seedling stage, set at 100%, to 117 in the young flag leaf. With the fairly resistant cultivar Vada the relative latent period increased from 123 to 233. The genotypic differences in latent period are very much greater in the adult plants stage than in the seedling stage. This and the fact that the relative latent period of the seedling does not predict the relative latent period of the adult plant very well, necessitates a cautious approach of seedling data.