Bovine Follicular Dynamics, Oocyte Recovery,and Development of Oocytes Microinjected with a Green Fluorescent Protein Construct

The present study was carried out to 1) evaluate the viability of in vitro fertilized zygotes after microinjection of DNA, 2) assess the influence of oocyte quality upon the development rate of embryos when injected with DNA, and 3) determine the integration frequency of green fluorescent protein DNA into microinjected embryos. Oocytes were aspirated from ovaries of nine nonlactating Holsteins and were categorized into grades A, B, C, and D. At 16 h after in vitro fertilization, approximately half of the pronuclear stage presumptive zygotes were classified as having 1 pronucleus or 2 pronuclei, and they were microinjected with DNA constructs. A potential predictor of DNA integration frequency at d 10 was assessment of the incidence of green fluorescing embryos. The proportion of cleaved embryos that developed to morulae or blastocysts was not different between groups with 1 pronucleus injected (45%), 1 pronucleus uninjected (64%), or 2 pronuclei injected (49%). However, the development of morulae or blastocysts was higher in the group with 2 pronuclei uninjected (69%). The overall developmental score of green fluorescent protein-positive embryos was higher for grade A oocytes (1.3 &#;&#;0.1) than for grade B (0.8 &#; 0.1), C (0.6 &#;&#;0.1), or D (0.3 &#;&#;0.1) oocytes. The results show that production of transgenic bovine blastocysts can occur from the microinjection of a presumptive zygote having only one visible pronucleus. Initial oocyte quality is an important factor in selection of oocytes suitable for microinjection of DNA and for preimplantation development to produce bovine transgenic embryos

Surfactant protein C peptides with salt-bridges (“ion-locks”) promote high surfactant activities by mimicking the α-helix and membrane topography of the native protein

Background. Surfactant protein C (SP-C; 35 residues) in lungs has a cationic N-terminal domain with two cysteines covalently linked to palmitoyls and a C-terminal region enriched in Val, Leu and Ile. Native SP-C shows high surface activity, due to SP-C inserting in the bilayer with its cationic N-terminus binding to the polar headgroup and its hydrophobic C-terminus embedded as a tilted, transmembrane α-helix. The palmitoylcysteines in SP-C act as ‘helical adjuvants’ to maintain activity by overriding the β-sheet propensities of the native sequences.Objective. We studied SP-C peptides lacking palmitoyls, but containing glutamate and lysine at 4-residue intervals, to assess whether SP-C peptides with salt-bridges (“ion-locks”) promote surface activity by mimicking the α-helix and membrane topography of native SP-C.Methods. SP-C mimics were synthesized that reproduce native sequences, but without palmitoyls (i.e., SP-Css or SP-Cff, with serines or phenylalanines replacing the two cysteines). Ion-lock SP-C molecules were prepared by incorporating single or double Glu−–Lys+ into the parent SP-C’s. The secondary structures of SP-C mimics were studied with Fourier transform infrared (FTIR) spectroscopy and PASTA, an algorithm that predicts β-sheet propensities based on the energies of the various β-sheet pairings. The membrane topography of SP-C mimics was investigated with orientated and hydrogen/deuterium (H/D) exchange FTIR, and also Membrane Protein Explorer (MPEx) hydropathy analysis. In vitro surface activity was determined using adsorption surface pressure isotherms and captive bubble surfactometry, and in vivo surface activity from lung function measures in a rabbit model of surfactant deficiency.Results. PASTA calculations predicted that the SP-Css and SP-Cff peptides should each form parallel β-sheet aggregates, with FTIR spectroscopy confirming high parallel β-sheet with ‘amyloid-like’ properties. The enhanced β-sheet properties for SP-Css and SP-Cff are likely responsible for their low surfactant activities in the in vitro and in vivo assays. Although standard 12C-FTIR study showed that the α-helicity of these SP-C sequences in lipids was uniformly increased with Glu−–Lys+ insertions, elevated surfactant activity was only selectively observed. Additional results from oriented and H/D exchange FTIR experiments indicated that the high surfactant activities depend on the SP-C ion-locks recapitulating both the α-helicity and the membrane topography of native SP-C. SP-Css ion-lock 1, an SP-Css with a salt-bridge for a Glu−–Lys+ ion-pair predicted from MPEx hydropathy calculations, demonstrated enhanced surfactant activity and a transmembrane helix simulating those of native SP-C.Conclusion. Highly active SP-C mimics were developed that replace the palmitoyls of SP-C with intrapeptide salt-bridges and represent a new class of synthetic surfactants with therapeutic interest

Reflections on a 'virtual' practice development unit: changing practice through identity development

Aims. This paper draws together the personal thoughts and critical reflections of key people involved in the establishment of a ‘virtual’ practice development unit of clinical nurse specialists in the south of England. Background. This practice development unit is ‘virtual’ in that it is not constrained by physical or specialty boundaries. It became the first group of Trust-wide clinical nurse specialists to be accredited in the UK as a practice development unit in 2004. Design and methods. The local university was asked to facilitate the accreditation process via 11 two-hour audio-recorded learning sessions. Critical reflections from practice development unit members, leaders and university staff were written 12 months after successful accreditation, and the framework of their content analysed. Findings and discussion. Practice development was seen as a way for the clinical nurse specialists to realize their potential for improving patient care by transforming care practice in a collaborative, interprofessional and evolutionary manner. The practice development unit provided a means for these nurses to analyse their role and function within the Trust. Roberts’ identity development model for nursing serves as a useful theoretical underpinning for the reflections contained in this paper. Conclusions. These narratives provide another example of nurses making the effort to shape and contribute to patient care through organizational redesign. This group of nurses began to realize that the structure of the practice development unit process provided them with the means to analyse their role and function within the organization and, as they reflected on this structure, their behaviour began to change. Relevance to clinical practice. Evidence from these reflections supports the view that practice development unit participants have secured a positive and professional identity and are, therefore, better able to improve the patient experience

Managing the risk of comparing estimated breeding values across flocks or herds through connectedness: a review and application

Comparing predicted breeding values (BV) among animals in different management units (e.g. flocks, herds) is challenging if units have different genetic means. Unbiased estimates of differences in BV may be obtained by assigning base animals to genetic groups according to their unit of origin, but units must be connected to estimate group effects. If many small groups exist, error of BV prediction may be increased. Alternatively, genetic groups can be excluded from the statistical model, which may bias BV predictions. If adequate genetic connections exist among units, bias is reduced. Several measures of connectedness have been proposed, but their relationships to potential bias in BV predictions are not well defined. This study compares alternative strategies to connect small units and assesses the ability of different connectedness statistics to quantify potential bias in BV prediction. Connections established using common sires across units were most effective in reducing bias. The coefficient of determination of the mean difference in predicted BV was a perfect indicator of potential bias remaining when comparing individuals in separate units. However, this measure is difficult to calculate; correlated measures such as prediction errors of differences in unit means and correlations among prediction errors are suggested as practical alternatives

Intraocular retinal transplants

Embryonic rat retinae transplanted into the anterior chamber of adult rat eyes of the same or different strain survive and grow. Light and electron microscopic studies show that the transplants undergo histogenetic differentiation, resulting in the development of mature inner and outer layer neurons and Muller glial cells. Vascular connections develop between the host iris and the retinal transplant. These initial observations indicate that retinal transplantation to a recipient eye is a procedure which offers ample opportunities for the study of problems related to neural development, retinal plasticity and repair

Association between FAMACHA scores and fecal egg counts in katahdin lambs

© 2017 American Society of Animal Science. All rights reserved. The FAMACHA system was introduced to the U.S. just over 10 yr ago to allow selective deworming of lambs with anemia associated with Haemonchus contortus and retard the development of anthelmintic resistance. The FAMACHA system was initially developed as a predictor of packed cell volume (PCV), but correlations between FAMACHA and fecal egg counts (FEC) have also been reported. It is important to understand factors that influence FAMACHA scores among farms to improve management of gastrointestinal nematodes. The objectives of this study were therefore to quantify associations between FAMACHA scores, FEC, BW, and age in Katahdin lambs at 2 different measurement times in 8 flocks in the eastern U.S., and to assess consistency of relationships between FAMACHA and FEC among flocks. Data came from 1,644 Katahdin lambs from 7 flocks sampled at approximately 90 d of age, and 1,295 lambs from 6 flocks sampled at approximately 120 d of age over a 5 yr period. Residual correlations among log-transformed FEC (LFEC), FAMACHA scores, BW, and lamb ages at each measurement time were determined. Repeatability of each variable was also determined as residual correlations among repeated measures. At both 90 and 120 d of age, correlations of FAMACHA scores with LFEC and BW were significant (P \u3c 0.001), but numerically modest (0.25 and -0.16, respectively at 90 d; 0.31 and -0.16, respectively at 120 d), demonstrating that higher FAMACHA scores were associated with higher FEC and more likely to be observed in lighter lambs. A small negative correlation was observed between FAMACHA score and lamb age (r = -0.05, P = 0.05, 90 d; r = -0.11, P \u3c 0.001, 120 d) indicating that younger lambs were more likely to have elevated FAMACHA scores. Thus, younger and lighter lambs will likely be more susceptible to parasitism and may need to be managed more diligently than older or heavier lambs. In addition, FAMACHA scores have potential to improve breeding value estimates in programs designed to genetically improve parasite resistance

Genetic impact of external Targhee sires at the U.S. Sheep Experiment Station: a case study of introgression

Sheep breeders requested that the U.S. Sheep Experiment Station (USSES) to participate in national genetic evaluation through the National Sheep Improvement Program (NSIP). The reasons included the need for (1) a comparison of the productivity of industry and United States Department of Agriculture (USDA) lines, (2) transparency of USDA flocks, (3) genetic ties for NSIP by sampling of industry flocks, and (4) development of premium genetic lines for public release. In response, USSES began to incorporate external sires from NSIP participating flocks into the USSES Targhee flock. Our objective, based on a pedigree analysis, was to test if introgression of external genetics into the flock was achieved. The pedigree included 13,189 animals with mean maximum generations, mean complete generations, and mean equivalent complete generations of 4.2, 1.8, and 2.6, respectively. The mean generation interval was 3.1 yr. The reference population was defined as lambs born from 2021 to 2023 (n = 792). Two additional populations were defined as the current mature ewe flock (n = 123) and the current mature rams (n = 14). The Genetic Conservation Index averaged 7.7 for the full population and 25.7 for the reference population. Overall inbreeding was 0.003 for the full population and 0.006 for the reference population. The rate of inbreeding was 0.0003 per generation. Average relatedness was 0.015 for the full population and 0.018 for the reference population. The effective number of founders, effective number of ancestors, and founder genome equivalents contributing to the reference population were 60, 39, and 19.1, respectively. The ratio of the effective number of founders to the effective number of ancestors was 1.5, indicating the presence of genetic bottlenecks. Measures of effective population size ranged from 102 to 547. Of the 704 offspring produced by external sires, 17 ram lambs and 132 ewe lambs were retained for breeding. The USSES sires produced 299 offspring with 2 ram lambs and 51 ewe lambs retained. Incorporating external sires resulted in a cumulative percentage of genetic variance of 48.8, 49.1, and 44.2 of external genetics for the reference population, current mature ewe flock, and current mature rams, respectively. Stakeholder needs were addressed by introgression of external sires and participation in NSIP, but future selection practices need to be modified to maintain a minimum of 50% USSES core genetics in the flock