Effect of ovine granulocyte-macrophage colony-stimulating factor on bovine in vitro embryo development and blastocyst interferon-τ secretion.

This experiment examined the effects of including recombinant ovine granulocyte-macrophage colony-stimulating factor (GMCSF) in in vitro culture on secretion of interferon-τ (IFNT) by bovine blastocysts. At 32 h post-insemination (p.i.), cleaved bovine zygotes were selected and incubated with or without GMCSF for either 48 h only (between 32 and 80 h p.i., Early) or until day 9 p.i. (Throughout). Concentrations of GMCSF (ng/ml) examined were as follows: Experiment 1: 2, 5, 10 and 50 (Early only); Experiment 2: 50 (Early and Throughout); Experiment 3: 2 and 10 (Early and Throughout). In none of the experiments did GMCSF have an effect (p > 0.05) on the numbers of blastocysts formed or blastocyst characteristics as assessed by cell number, proportion of apoptotic cells or oxidation of pyruvate. When GMCSF was included in culture medium between 32 and 80 h p.i. (Early), IFNT concentrations were lower (in media drops recovered after culture of groups of embryos for 48 h between days 7 and 9 p.i. and normalized by the numbers of blastocysts developing within each drop) compared to no inclusion of GMCSF or GMCSF present Throughout culture (Experiment 2, p > 0.05; Experiment 3, p = 0.038). IFNT was present in media drops in which groups of embryos had been incubated between days 7 and 9 p.i. but in which no blastocysts had developed. Experimental treatment did not influence (p > 0.05) IFNT secretion by blastocysts incubated individually for 24 h. However, during the 24-h individual culture, blastocysts recovered on day 7 secreted less IFNT than blastocysts recovered on day 8 (mean ± SE; 15 ± 1.3 v 30 ± 3.6 pg/ml; p < 0.001). In conclusion, in contrast to previous studies in the ovine, GMCSF did not increase IFNT secretion but in agreement with the ovine did not affect bovine blastocyst development.

Dietary carbohydrates and amino acids influence oocyte quality in dairy heifers.

The objective of the present experiment was to determine whether increasing plasma insulin by different nutritional regimes affects oocyte quality. Holstein dairy heifers (eight per treatment) were assigned, using a two times two factorial design, to diets containing either low or high dietary leucine and either low or high dietary starch. Each heifer underwent six sessions of ovum pick-up beginning 25 days after introduction of the diets. Oocyte quality was assessed by development to the blastocyst stage in synthetic oviducal fluid following in vitro fertilisation. Feeding diets containing high leucine resulted in significantly higher plasma free leucine and tyrosine concentrations. The high-starch diet significantly increased plasma insulin but not glucagon concentration, whereas high dietary leucine increased plasma glucagon but not insulin. Oocyte cleavage was not influenced by diet. The high-starch diet, which was associated with a high plasma insulin : glucagon ratio, had adverse effects on oocyte quality that were avoided when leucine intake was increased. There was an association between total plasma free amino acid concentration and oocyte cleavage. Therefore, in dairy heifers dietary amino acids and carbohydrates during antral follicle development appear to mediate effects on oocyte quality by different mechanisms. These findings have implications for both diet formulation and feeding regimes.

Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep.

Nuclear transfer offers a cell-based route for producing precise genetic modifications in a range of animal species. Using sheep, we report reproducible targeted gene deletion at two independent loci in fetal fibro-blasts. Vital regions were deleted from the alpha(1,3)galactosyl transferase (GGTA1) gene, which may account for the hyperacute rejection of xenografted organs, and from the prion protein (PrP) gene, which is directly associated with spongiform encephalopathies in humans and animals. Reconstructed embryos were prepared using cultures of targeted or nontargeted donor cells. Eight pregnancies were maintained to term and four PrP-/+ lambs were born. Although three of these perished soon after birth, one survived for 12 days. These data show that lambs carrying targeted gene deletions can be generated by nuclear transfer.

Feeding frequency has diet-dependent effects on plasma hormone concentrations but does not affect oocyte quality in dairy heifers fed fibre- or starch-based diets.

The post-fertilisation developmental capacity of bovine oocytes recovered by ultrasound guided transvaginal follicular aspiration (ovum pick-up, OPU) is influenced by diet-induced changes in hormone and metabolite concentrations. The objectives of this experiment were first to determine whether post-prandial changes in hormone concentrations, induced by changing the frequency of feeding, influenced oocyte quality and second whether changes in plasma glucagon concentration were associated with oocyte quality. Using a 2 × 2 factorial design, Holstein heifers (six per treatment) were fed either fibre- or starch-based diets containing either 189 or 478 g starch/kg dry matter. The diets were offered in either two or four equal meals per day and supplied twice the maintenance energy requirement. Blood samples were obtained both at weekly intervals (three samples per heifer, collected before feeding) during the experiment and throughout an entire 24-h period (15 or 17 samples per heifer for twice or four times daily-fed heifers, respectively). Each heifer underwent six sessions of OPU (twice weekly) beginning 25 days after introduction of the diets. Oocyte quality was assessed by development to the blastocyst stage in synthetic oviductal fluid following in vitro fertilisation. Mean weekly plasma insulin concentrations did not differ between diets, but plasma glucagon concentrations were greatest when heifers were fed the starch-based diet twice daily compared with the other diets. When heifers were offered four meals per day, there were no meal-related changes in hormone concentrations. However, when heifers were offered two meals per day, plasma insulin concentration increased after feeding the starch-based, but not the fibre-based diet. Plasma glucagon concentration increased after meals when heifers were fed twice daily and the increase was substantially greater when the starch-based diet was fed. Treatments did not influence (overall mean with mean ± s.e.) ovarian follicle size distribution or oocyte recovery by OPU (6.2 ± 0.4 per heifer), the proportion of oocytes that cleaved following insemination (0.57 ± 0.030) or blastocyst yield (0.27 ± 0.027 of oocytes cleaved). In conclusion, by feeding diets differing in carbohydrate source at different frequencies of feeding, meal-related changes in plasma hormone profiles were altered significantly, but oocyte quality was not affected. Therefore effects of diet on oocyte quality appear not to be mediated by meal-related fluctuations in hormone concentrations.

The RPM1 plant disease resistance gene facilitates a rapid and sustained increase in cytosolic calcium that is necessary for the oxidative burst and hypersensitive cell death.

Early events occurring during the hypersensitive resistance response (HR) were examined using the avrRpm1/RPM1 gene-for-gene interaction in Arabidopsis challenged by Pseudomonas syringae pv. tomato. Increases in cytosolic Ca2+ were measured in whole leaves using aequorin-mediated bioluminescence. During the HR a sustained increase in Ca2+ was observed which was dependent on the presence of both a functional RPM1 gene product and delivery of the cognate avirulence gene product AvrRpm1. The sequence-unrelated avirulence gene avrB, which also interacts with RPM1, generated a significantly later but similarly prolonged increase in cytosolic Ca2+. Accumulation of H2O2 at reaction sites, as revealed by electron microscopy, occurred within the same time frame as the changes in cytosolic Ca2+. The NADPH oxidase inhibitor diphenylene iodonium chloride did not affect the calcium signature, but did block H2O2 accumulation and the HR. By contrast, the calcium-channel blocker LaCl3 suppressed the increase in cytosolic Ca2+ as well as H2O2 accumulation and the HR, placing calcium elevation upstream of the oxidative burst.

Survey of the late consequences of polio in Edinburgh and the Lothians.

OBJECTIVE: This study aimed to identify the nature and frequency of new symptoms and associated disabilities experienced by people who suffered poliomyelitis many years ago, and their access to hospital services. DESIGN: Postal survey regarding symptoms, disability (Office of Population Censuses & Surveys (OPCS) disability questionnaire), and experience of services. SUBJECTS & SETTING: People known to the British Polio Fellowship and the regional rehabilitation service who had previously had polio and were resident in Edinburgh and the Lothians. RESULTS: The study population of 125 people had a median age of 59 years, and 60% were female. The most common symptoms, present in over 60%, were: cold intolerance, fatigue, increased and new muscle weakness, muscle and joint pain, and sleep disturbance. Motor disabilities, particularly of lower limbs, were prominent. Patient contact with hospital services in the preceding five years was: clinic (62%); physiotherapy (46%); occupational therapy (29%); speech therapy (2%); wheelchair service (42%); orthotics (40%). CONCLUSIONS: New problems are sufficiently frequent and severe to warrant a more detailed study to quantify the health care requirements of the post-polio population.

Cloned mice derived from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation.

Our objective was to induce enucleation (IE) of activated mouse oocytes to yield cytoplasts capable of supporting development following nuclear transfer. Fluorescence microscopy for microtubules, microfilaments, and DNA was used to evaluate meiotic resumption after ethanol activation and the effect of subsequent transient treatments with 0.4 micro g/ml of demecolcine. Using oocytes from B6D2F1 (C57BL/6 x DBA/2) donors, the success of IE of chromatin into polar bodies (PBs) was dependent on the duration of demecolcine treatment and the time that such treatment was initiated after activation. Similarly, variations in demecolcine treatment altered the proportions of oocytes exhibiting a reversible compartmentalization of chromatin into PBs. Treatment for 15 min begun immediately after activation yielded an optimized IE rate of 21% (n = 80) when oocytes were evaluated after overnight recovery in culture. With this protocol, 30-50% of oocytes were routinely scored as compartmentalized when assessed 90 min postactivation. No oocytes could be scored as such following overnight recovery, with 66% of treated oocytes cleaving to the 2-cell stage (n = 80). Activated cytoplasts were prepared by mechanical removal of PBs from oocytes whose chromatin had undergone IE or compartmentalization. These cytoplasts were compared with mechanically enucleated, metaphase (M) II cytoplasts whose activation was delayed in nuclear transfer experiments using HM-1 embryonic stem cells. Using oocytes from either B6D2F1 or B6CBAF1 (C57BL/6 x CBA) donors, the in vitro development of cloned embryos using activated cytoplasts was consistently inferior to that observed using MII cytoplasts. Live offspring were derived from both oocyte strains using the latter, whereas a single living mouse was cloned from activated B6CBAF1 cytoplasts.