Cell Cycle Stage Analysis of Rabbit Foetal Fibroblasts and Cumulus Cells

Synchronization of the cell cycle stages in G0/G1 phase is one of the key factors determining the success of nuclear transplantation. Serum deprivation, contact inhibition and chemical inhibitors are widely used methods for this purpose. In this study, cell cycle stages of foetal fibroblasts and cumulus cells were determined using flow cytometry [fluorescence-activated cell scan (FACS)]. Foetal fibroblasts (in vitro cultured for 72-120 h) and fresh cumulus cells were analysed in Experiment 1. Fifty to 55% proliferating fibroblasts remained in G0/G1 phase compared with 78% in confluent culture (p <0.05). In contrast to foetal fibroblasts, fresh cumulus cells maintained 90% of the population in the G0/G1 stage. When serum was retrieved from the proliferating fibroblasts from day 1 to day 5 (Experiment 2), proportions of G0/G1 cells increased from the initial ratio of 53 to 87% at day 4 of starvation, which was significantly higher than the non-starved proliferating cells (p <0.05). In Experiment 3, fibroblasts were treated with aphidicolin (0.1 microg/ml, 6 h), demicolcine (0.5 microg/ml, 10 h), or a combination of these two chemicals to synchronize the cell cycle stages. Surprisingly, no differences or significantly lower in the proportions of G0/G1)phase cells were detected (25-50%) compared with the uncontrolled growing cells (53%). These results suggested that fresh cumulus cells rest their cell cycle in G0/G1 stage. Serum deprivation became effective in the first 24 h and reached the highest proportions during days 4-5 after deprivation. Chemical synchronization of the cell cycle stage of rabbit foetal fibroblasts to G0/G1 phase appeared less effective compared to serum deprivation.     

Development of an indirect ELISA for detection of antibody to wobbly possum disease virus in archival sera of Australian brushtail possums (Trichosurus vulpecula) in New Zealand

AIMS: To develop an indirect ELISA based on recombinant nucleocapsid (rN) protein of wobbly possum disease (WPD) virus for investigation of the presence of WPD virus in Australian brushtail possums (Trichosurus vulpecula) in New Zealand.

METHODS: Pre- and post-infection sera (n=15 and 16, respectively) obtained from a previous experimental challenge study were used for ELISA development. Sera were characterised as positive or negative for antibody to WPD virus based on western-blot using WPD virus rN protein as antigen. An additional 215 archival serum samples, collected between 2000–2016 from five different regions of New Zealand, were also tested using the ELISA. Bayesian modelling of corrected optical density at 450?nm (OD₄₅₀) results from the ELISA was used to obtain estimates of receiver operating characteristic (ROC) curves to establish cut-off values for the ELISA, and to estimate the prevalence of antibody to WPD virus.

RESULTS: Western blot analysis showed 5/14 (36%) pre-infection sera and 11/11 (100%) post-infection sera from experimentally infected possums were positive for antibodies to WPD virus. Bayesian estimates of the ROC curves established cut-off values of OD₄₅₀≥0.41 for samples positive, and OD₄₅₀<0.28 for samples negative for antibody to WPD virus, for sera diluted 1:100 for the ELISA. Based on the model, the estimated proportion of samples with antibodies to WPD virus was 0.30 (95% probability interval=0.196–0.418). Of the 230 archival serum samples tested using the ELISA, 48 (20.9%) were positive for antibody to WPD virus, 155 (67.4%) were negative and 27 (11.7%) equivocal, using the established cut-off values. The proportion of samples positive for WPD virus antibody differed between geographical regions (p<0.001).

CONCLUSION: The results suggested that WPD virus or a related virus has circulated among possums in New Zealand with differences in the proportion of antibody-positive samples from different geographical regions. Antibodies to WPD virus did not seem to protect possums from disease following experimental infection, as one third of possums from the previous challenge study showed evidence of pre-existing antibody at the time of challenge. These results provide further support for existence of different pathotypes of WPD virus, but the exact determinants of protection against WPD and epidemiology of infection in various regions of New Zealand remain to be established.

CLINICAL RELEVANCE: Availability of the indirect ELISA for detection of WPD virus antibody will facilitate prospective epidemiological investigation of WPD virus circulation in wild possum populations in New Zealand.     

Vertical Flux of Biogenic Carbon in the Ocean: Is There Food Web Control?

Models of biogenic carbon (BC) flux assume that short herbivorous food chains lead to high export, whereas complex microbial or omnivorous food webs lead to recycling and low export, and that export of BC from the euphotic zone equals new production (NP). In the Gulf of St. Lawrence, particulate organic carbon fluxes were similar during the spring phytoplankton bloom, when herbivory dominated, and during nonbloom conditions, when microbial and omnivorous food webs dominated. In contrast, NP was 1.2 to 161 times greater during the bloom than after it. Thus, neither food web structure nor NP can predict the magnitude or patterns of BC export, particularly on time scales over which the ocean is in nonequilibrium conditions.     

Associations between management practices and colostrum quality on New Zealand dairy farms

Abstract

AIMS: To describe colostrum quality in spring-calving dairy herds in New Zealand, in terms of Brix, pH and total and coliform bacterial counts and to investigate associations between farm management practices and these measures of colostrum quality.

METHODS: In June 2015, commercial dairy farms (n=105), located in North and South Islands of New Zealand, were visited shortly after the first cows had calved, and when approximately 50% and 80% of the herd had calved (early, middle and late visits). One litre of pooled colostrum that was being fed to newborn calves was collected at each visit and used to determine Brix, pH, total bacterial and coliform counts. A survey of calf management practices was conducted with the herd manager or calf rearer after the final visit.

RESULTS: Of 298 pooled colostrum samples tested 29/298 (9.7%) had Brix >22%. Brix was higher on farms where calves were picked up twice daily compared with once daily (18.2 (95% CI=16.5–19.9)% vs. 15.9% (95% CI=15.2–16.6)%; p=0.012), and was lower where first milking colostrum was combined with colostrum obtained at later milkings (15.0 (95% CI=13.9–16.1)%) compared with where it was not (16.9 (95% CI=16.3–17.6)%; p=0.002). Vaccination of all cows against calf diarrhoeal pathogens was associated with increased Brix compared with no vaccination (18.1 (95% CI=16.6–19.6)% vs. 16.3 (95% CI=15.6–17.0)%; p=0.033). Mean pH of samples tested decreased from 5.97 (95% CI=5.84–6.09) to 5.58 (95% CI=5.45–5.71) for early and late-season visits, respectively (p<0.001). Of 268 samples tested, 23 (8.6%) had bacterial counts below the recommended threshold of 1.00×10⁵ cfu/mL. Mean bacterial counts increased from 2.75 (95% CI=1.80–3.70)×10⁸ to 4.99 (95% CI=3.95–6.03)×10⁸ cfu/mL for early and late-season visits, respectively (p<0.001). Of 259 samples tested, 23 (8.9%) had coliform counts below the recommended threshold of 1.00×10⁴ cfu/mL.

CONCLUSIONS AND CLINICAL RELEVANCE: On a large majority of dairy farms included in this study the pooled colostrum fed to newborn calves had sub-optimal Brix and excessive bacterial counts. Farm-level risk factors such as twice daily pick up of calves from the paddocks, herd vaccination and feeding pooled colostrum that did not combine colostrum obtained at later milkings with first milking colostrum were associated with colostrum quality measures.     

Modular hybrid total hip arthroplasty. Experimental study in dogs

Background

This prospective experimental study evaluated the surgical procedure and results of modular hybrid total hip arthroplasty in dogs.

Methods

Ten skeletally mature healthy mongrel dogs with weights varying between 19 and 27 kg were used. Cemented modular femoral stems and uncemented porous-coated acetabular cups were employed. Clinical and radiographic evaluations were performed before surgery and at 30, 60, 90, 120, 180 and 360 days post-operation.

Results

Excellent weight bearing was noticed in the operated limb in seven dogs. Dislocation followed by loosening of the prosthesis was noticed in two dogs, which were therefore properly treated with a femoral head osteotomy. Femoral fracture occurred in one dog, which was promptly treated with full implant removal and femoral osteosynthesis.

Conclusions

The canine modular hybrid total hip arthroplasty provided excellent functionality of the operated limb.     

A finite element model of the bovine claw under static load for evaluation of different flooring conditions

AIM: To create a computer-based finite element (FE) model of the bovine claw and to use finite element analysis (FEA) to estimate stress and deformation of a physiologically-shaped model claw under static load, to visualise potential material weakness and to evaluate the effect of different flooring conditions.

METHODS: Model geometry was derived using digitalised images from a recently trimmed, sound, hind claw from a 4-year-old Austrian Fleckvieh cow. Material properties of bovine claw horn were defined from preliminary investigations and recently established material data, using a modulus of elasticity from 200 to 600 N/mm² . Meshing of the model was performed with 42,127 elements based on 116,141 nodes. Loading of the model was defined at 756 N per claw on a hard and soft surface.

RESULTS: The FE model of the bovine claw under a load of 756 N showed only minimal deformation, most of which took place at the axial wall. Highest stresses were evident in the proximal axial wall, the outer edge of the weight-bearing surface and under the heels. The claw-floor contact image showed a pressure distribution resembling the distal rim of the claw wall. On the hard surface, the maximum stresses were three times higher than those on the soft surface.

CONCLUSIONS AND CLINICAL RELEVANCE: FEA allowed visualisation of the effects that loading on different floor surfaces have on the biomechanics of the claw. Uneven preparation of the claw sole resulted in high stresses at and close to irregularities of the sole. Consequences were more severe on harder flooring. The model supports the hypothesis that mechanical factors play a substantial role in the pathogenesis of claw lesions.