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.     

Adverse drug reactions: Report of the Australian Veterinary Association Adverse Drug Reaction Subcommittee, 1994

Seventy-seven reports of suspected adverse drug reactions (ADRs) were received by the Adverse Drug Reaction Subcommittee (ADRSc) of the Australian Veterinary Association from April 1993 to December 1994 inclusive. The number of reports received/number of animals involved per species were: dogs (32/44), cats (18/31), horses (17/48), and cattle (10/21). Of these, 49 (64%) were classified as definite ADRs and 9 (12%) as probable ADRs. In 11 (14%) reports an ADR could not be substantiated or there was insufficient information available to make a decision. Eight reports were not classified because the manufacturer and the ADRSc disagreed as to the appropriate classification. Sixteen reports involved apparent hypersensitivity reactions, which resulted in death on 6 occasions. Six reports were associated with ‘off label’ use and 1 report with use of an expired product. Of the definite, probable and unclassified reports of suspect ADRs, the most frequent types of drugs involved were antimicrobial drugs (13 reports), anthelmintics (13), insecticides (11), vaccines (10), nonsteroidal anti-inflammatory preparations (5), chondroprotective agents (4), anaesthetic/sedative agents (4) and vitamin preparations (2). Single reports concerning definite, probable or unclassified ADRs to a vasodilator, a corticosteroid, a local anaesthetic and a disinfectant were received.     

The diagnosis and treatment of a thoracic abscess in an alpaca

A 6-month-old alpaca presented for lethargy, failure to thrive and leucocytosis. Diagnostic procedures, including thoracic ultrasound, radiographs and a computed tomography scan, were used to document a thoracic mass. Based on appearance and ultrasound-guided aspiration of purulent material, the mass was determined to be a large abscess. The abscess was treated with surgical drainage and long-term antibiotic therapy. The origin of this thoracic abscess was felt to be the caudal mediastinum, secondary to bacterial seeding of the caudal mediastinal lymph nodes. Although an aetiological agent was not definitively determined, the most likely was Actinomyces spp or Arcanobacterium pyogenes. The alpaca made a complete recovery following treatment.     

Crystalline Ropes of Metallic Carbon Nanotubes

Fullerene single-wall nanotubes (SWNTs) were produced in yields of more than 70 percent by condensation of a laser-vaporized carbon-nickel-cobalt mixture at 1200degreesC. X-ray diffraction and electron microscopy showed that these SWNTs are nearly uniform in diameter and that they self-organize into "ropes," which consist of 100 to 500 SWNTs in a two-dimensional triangular lattice with a lattice constant of 17 angstroms. The x-ray form factor is consistent with that of uniformly charged cylinders 13.8 +/- 0.2 angstroms in diameter. The ropes were metallic, with a single-rope resistivity of <10(-4) ohm-centimeters at 300 kelvin. The uniformity of SWNT diameter is attributed to the efficient annealing of an initial fullerene tubelet kept open by a few metal atoms; the optimum diameter is determined by competition between the strain energy of curvature of the graphene sheet and the dangling-bond energy of the open edge, where growth occurs. These factors strongly favor the metallic (10,10) tube with C5v symmetry and an open edge stabilized by triple bonds.     

Morphological Changes of the Endometrial Epithelium in the Bitch during Metoestrus and Anoestrus

Although cyclic changes of the endometrium in dogs involving both stromal and glandular compartments have been described, the fate of the surface epithelium after progressive growth and secretion is still unclear. In the present study, uteri of 43 healthy bitches in metoestrus and anoestrus were examined macroscopically and histologically. Tissue biopsies were taken from three different locations (cranial and middle parts of uterine horns and bifurcation). The stage of the oestrous cycle was determined by evaluation of progesterone and oestradiol levels in plasma hormone and was also confirmed clinically. Crypts formed in the luteal phase were covered with a columnar epithelium which gradually underwent fatty degeneration. In addition, the stromal part of the crypts disappeared and finally, in early anoestrus, epithelial sheaths desquamated and shed off into the uterine lumen. The surface epithelium was replaced by new cuboidal cells proliferating and migrating from the glandular openings. These findings were confirmed by oil red O staining and immunohistochemical detection of proliferation with Ki‐67 marker.     

Inhalant anesthetic requirement in cats is animal-dependent

Minimum alveolar concentration (MAC) of an inhalant is an indicator of its anesthetic potency. Individuals vary in their sensitivity to anesthetic agents as demonstrated by different individual MAC values. We hypothesized that individual animal sensitivity would be maintained with different inhalant anesthetics. As part of separate studies, six female DSH cats, aged 24 ± 2.5 (mean ± SD) months and weighing 3.5 ± 0.3 kg, were studied similarly on three separate occasions over a 12‐month period to determine the MAC of isoflurane (ISO), sevoflurane (SEVO), and desflurane (DES), respectively. In each study, chamber induction was followed by orotracheal intubation, and anesthesia was maintained via a nonrebreathing circuit. ECG, pulse oximetry, Doppler systolic blood pressure, end‐tidal gases, and esophageal temperature were monitored. End‐tidal gases were hand‐sampled from a catheter whose tip lay level with the distal end of the ET tube. Gases were analyzed by Raman spectrometry and, for each agent, the analyzer was calibrated with at least three gas standards. MAC was determined in triplicate using standard tail‐clamp technique. Data were analyzed by two‐way anova followed by Tukey's test and significant differences were found. Average MACs (%) for ISO, SEVO, and DES were 1.90 ± 0.18, 3.41 ± 0.65, and 10.27 ± 1.06, respectively. Body temperatures, Doppler systolic blood pressure, and SpO₂ were recorded at the time of MAC determinations for ISO, SEVO, and DES were 38.3 ± 0.3, 38.6 ± 0.1, 38.3 ± 0.35 °C; 71 ± 8, 75 ± 16, 88 ± 12 mm Hg; 99 ± 1, 99 ± 1, 99 ± 1%, respectively. Both the anesthetic agent and the individual cat had significant effects on MAC (p = 0.0001 and 0.0185, respectively). MAC varied between individuals and cats were consistent in their order of sensitivity to inhalant anesthetics across the three agents. Within this group of cats, the relationship of individual MAC to the group MAC for each of the three inhalant agents was maintained. This suggests that any individual may be consistently more or less sensitive to a variety of inhalant agents.     

VCARS – veterinary computerized anesthetic record system

VCARS, the Veterinary Computerized Anesthetic Record System, has been developed to capture pre-, peri- and post-anesthetic data using Tablet PCs, 802.11b wireless networks, and a web-based database. Patient demographics, anticipated procedures and anesthetist, service information and hematologic and chemistry values are imported from the Veterinary Medical Teaching Hospital patient information system. Using a wireless Tablet PC, pre-anesthetic examination findings are recorded and an anesthetic plan including anesthetic drugs and anesthetic and monitoring equipment is developed. During induction and maintenance of anesthesia, physiologic variables, drug, fluid, anesthetic gas and oxygen administration, laboratory values, patient location, and important events can be charted with the simplicity of a paper anesthetic record. This information can be manipulated for display in a variety of ways depending on the specific needs of the case. Tools for calculating optimal fluid rates and drug dosages are incorporated into the design. The anesthetic records from multiple cases can be viewed simultaneously using a centrally-located monitor. Detailed audit trails ensure data integrity. A high-end search engine will allow rapid and complete retrieval of patient and anesthetic information. Macromedia flash is used to allow temporary disconnection from the wireless network without losing the ability to view, add, or edit data. The initial stages of software development are nearing completion, a wireless network is in place and hardware is being purchased. A pilot study will be conducted using manual entry of physiologic data prior to integration of automatically captured patient physiological variables. It is anticipated that this system will drastically improve the accuracy of data collection and retrieval and will provide important information about anesthetic management allowing improvement in overall patient care.     

Infestation in the dog by the paralysis tick, Ixodes holocyclus — 5. Treatment

In this study the value of drugs administered with hyperimmune serum in the treatment of advanced disease produced by Ixodes holocyclus was compared under controlled conditions. All control dogs died rapidly whereas one dog survived and 3 dogs died after receiving hyperimmune serum alone. When promethazine hydrochloride was administered with hyperimmune serum 2 dogs recovered rapidly while the remaining 2 died. Administration of dexamethasone and hyperimmune serum allowed 3 dogs to survive while administration of phenoxybenzamine hydrochloride in conjunction with hyperimmune serum allowed rapid recovery of all 4 dogs. Phenoxybenzamine hydrochloride, an alpha-adrenergic blocking drug, was chosen because of its potential to attenuate the arterial hypertension previously reported (Ilkiw et al 1988). The survival of all dogs together with the rapid return to normality indicated that this drug was beneficial in the treatment of dogs with advanced signs of tick paralysis.     

Heliotropium europaeum poisoning of sheep with low liver copper concentrations and the preventive efficacy of cobalt and antimethanogen

In a field experiment in the Mallee district of Victoria, Merlno xBorder Leicester ewes and wethers grazed Heliotropium europaeum (heliotrope) over periods of 3 to 4 months in 4 successive years. By the end of the second year 12% (14 of 120) of the sheep had died; after 4 years the loss attributable to heliotrope was between 18% and 35%. Mortality was not affected by intraruminal treatment with cobalt or antimethanogen. At the end of the experiment the highest concentration of copper in the liver was 1.95 mmol/kg wet weight (approximately 413 micrograms/g dry weight). The relatively low mortality from primary heliotrope poisoning and the low concentration of copper in the liver of sheep grazing the plant are discussed in relation to the contrasting situation that prevails in the Riverina area of New South Wales. The importance of local environmental factors in the management of heliotrope grazing by sheep is emphasised, particularly in relation to the number of seasons in which the plant may be a major component of the diet.     

Neuronal Responses in the Brainstem and Hypothalamic Nuclei Following Insulin Treatment During the Late Follicular Phase in the Ewe

The aim of this study was to determine the neuronal responses following insulin administration during the late follicular phase. Intact ewes were given either saline or insulin (5 IU/kg, i.v.) at 35 h after progesterone withdrawal and killed 3 h later. There was a marked increase in the number of Fos‐positive noradrenergic neurones in the caudal brainstem of insulin‐treated ewes. In the hypothalamic paraventricular nucleus, insulin treatment increased the presence of Fos‐positive corticotrophin‐releasing hormone neurones (from 2% to 98%) and Fos‐positive arginine vasopressin parvocellular neurones (from 2% to 46%). Interestingly, after insulin treatment, despite a general increase in Fos‐positive neurones in the arcuate nucleus (ARC), there was a marked reduction (from 47% to 1%) in Fos‐positive β‐endorphin neurones. Similarly, colocalized Fos and oestradiol receptor (ER) α‐positive neurones decreased in the ARC after insulin (from 7% to 3%). Conversely, in the ventromedial nucleus, ERα‐positive neurones with Fos increased (from 7% to 22%) alongside a general increase in Fos‐positive neurones. Overall, a complex system of neurones in brainstem and hypothalamus is activated following insulin administration during the late follicular phase.