The use of vascular access ports in nonanesthetized green iguanas (iguana iguana) to collect baseline arterial blood gas parameters.

The green iguana, Iguana iguana, is used as a model in reptile anesthesia research because of its size, availability, and the body of knowledge characterizing its physiology. Arterial blood gas values in nonanesthetized green iguanas have not been determined because of the technical difficulty involved. Vascular access port (VAP) placement to facilitate blood sampling has been described in other species, but not lacertilians. This abstract describes the technique for placement of VAPs and the values for arterial blood gas parameters in seven 1 kg adult green iguanas. Using sterile technique, a 1.5 cm incision was made on the lateral side of the neck. Blunt dissection ventral to the external jugular vein revealed the internal and external carotid arteries near their bifurcation. The catheter was inserted into the internal carotid artery and then guided to the common carotid artery. The other end of the catheter was tunneled below the skin to a subcutaneous location, caudal‐dorsal to the iSPSilateral scapula. The skin was closed and the port was flushed twice a week with heparinized saline. Post‐operatively, the VAPs were well tolerated by the iguanas. Difficulties included port disconnection (n = 1), inability to aspirate blood after a few weeks (n = 2), and infection (n = 1). The iguanas were breathing room air prior to and during blood collection. From the five functional VAPs, the blood pH, PCO₂, PO₂, HCO^‐₃, and BE (measured at 37 °C) were 7.45 ± 0.06; 37.5 ± 7.0 mm Hg, 99.0 ±16.6 mm Hg, 25.4 ± 2.5 mmol L^–1, and 1.5 ±2.4 mmol L^–1 respectively (mean ± SD). VAPs can be successfully used to facilitate collection of arterial blood gas samples in green iguanas. These values are similar to those reported for most mammalian species. This technique should facilitate research in anesthesiology and respiratory physiology of iguanas and other lacertilians.     

Breeding Soundness Evaluation and Semen Analysis for Predicting Bull Fertility

Bull fertility is influenced by numerous factors. Although 20–40% of bulls in an unselected population may have reduced fertility, few are completely sterile. Breeding soundness refers to a bull's ability to get cows pregnant. A standard breeding soundness evaluation identifies bulls with substantial deficits in fertility, but does not consistently identify sub-fertile bulls. In this regard, the use of frozen-thawed semen (from bulls in commercial AI centres) that meets minimum quality standards can result in pregnancy rates that differ by 20–25 percentage points. Although no single diagnostic test can accurately predict variations in fertility among bulls that are producing apparently normal semen, recent studies suggested that a combination of laboratory tests were predictive of fertility. This review is focused on recent developments in prediction of bull fertility, based on assessments at the molecular, cellular and whole-animal levels.     

Resistance to synthetic pyrethroid pour-on insecticides in strains of the sheep body louse Bovicola (Damalinia) ovis

Groups of sheep infested with strains of Bovicola (Damalinia) ovis were obtained from flocks either with a history of failure to control lice with synthetic pyrethroid (SP) pour-on insecticides, or from farms where SP compounds were not used. The sheep were treated according to the manufacturer's recommendations with registered "off-shears" SP formulations. All treatments were applied under ideal conditions with doses calculated on an individual body weight basis and applied to the dorsal mid-line from the base of the neck to the butt of the tail. Treated sheep were kept in pens and maintained in separate groups. The pour-on SP treatments significantly reduced the lice population but failed to eliminate the infestation in 7 of 13 experiments in sheep carrying strains of lice with resistance factors of greater than 4 to at least one of the SP compounds. Failures occurred with all three of the SP pour-ons currently registered for lice control in NSW and with both water-based and organic solvent-based formulations.     

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.     

Buffalo (Bubalus bubali) Late Embryo and Foetus Development: A Morphological Analysis

Many researches describe the embryonic developmental features in domestic animals; however, in farm animals, they are scarce. Most farm animal studies are related to assisted reproduction and embryos transfer techniques. But, morphological features and size measure to estimate the age gestation are rarely reported in literature. Thus, in this study, we described the developmental changes in the bubaline (Bubalus bubali) concepts from 21 to 60 days of gestation. Our results revealed that buffalo embryos similar morphological characteristics similar to other mammalian species. Also, similarities between bovine and bubaline persist; except on foetal stages when buffalos have a faster development than bovine. Therefore, buffalo's gestation period exhibits some varieties and accurate embryo age is more difficult. Yet, when we use a combination of the crown–rump, macroscopic analysis and alizarin red, it is possible to describe better the whole embryogenesis stages of the buffalo and which can contribute for future reproduction researches and applications in veterinary practice.     

Pathophysiology of humeral fractures in a sample of dairy heifers

AIMS: To investigate the pathophysiology of humeral fractures in first-lactation dairy heifers in the North Island of New Zealand.

METHODS: Ten 2-year-old dairy heifers with humeral fractures were subject to euthanasia and the fractured and non-fractured contralateral humeri were collected. Humeri were also collected from 10 unaffected 2-year-old dairy heifers sent for slaughter. Humeri from heifers with and without fractures were examined using computed tomography (CT), and four slices of the diaphysis and lower metaphysis (D1–4) were analysed using the Bone J plug-in for Image J. The humeri were sectioned sagittally and 5?mm bone slabs were processed for histopathology.

RESULTS: There were no differences in bone length between the humeri from heifers with or without fractures (p=0.31). Median cortical bone mineral density at D1 was increased in humeri from affected compared with unaffected heifers (810 vs. 783?mg/cm³; p=0.03), cortical area at D1 was reduced (816 vs. 1,037?mm²; p=0.04), the median stress strain index, a calculated theoretical measure of bone strength, at D1 was decreased (7,288 vs. 9,072?mm³; p<0.01), and the median ratio of overall bone volume (BV) to total volume (TV) was decreased (0.32 vs. 0.38; p<0.01). The median periosteal circumference at D1 was also reduced in humeri from affected compared with unaffected heifers (151 vs. 173?mm; p<0.01). Using a binary logistic regression model, BV/TV was the only variable associated with humeral fractures (p=0.03).In nine of 10 fractured humeri the fracture appeared to have started just distal to the head of the humerus and spiralled distally down the diaphysis to end just above the humeral condyles. Histopathological findings included a reduction in the number, and thickness, of trabeculae in the metaphysis; metaphyseal growth arrest lines, and osteoclastic resorption in fractured humeri. Concentrations of copper in serum from four of five animals with fractures were within, and one was below, normal reference ranges, while concentration of copper in the livers of three heifers with fractures were below adequate ranges.

CONCLUSIONS: The CT and histological findings were consistent with a diagnosis of osteoporosis. We propose that humeral fractures in dairy heifers are associated with osteoporosis, possibly as a result of insufficient deposition of bone during growth because of protein-calorie malnutrition. Increased osteoclastic resorption of bone associated with calcium mobilisation for lactation, and periods of copper deficiency may contribute to bone weakening.     

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.