Derivation of Canine Induced Pluripotent Stem Cells

Dogs and humans have many inherited genetic diseases in common and conditions that are increasingly prevalent in humans also occur naturally in dogs. The use of dogs for the experimental and clinical testing of stem cell and regenerative medicine products would benefit canine health and welfare and provide relevant animal models for the translation of therapies to the human field. Induced pluripotent stem cells (iPSCs) have the capacity to turn into all cells of the body and therefore have the potential to provide cells for therapeutic use and for disease modelling. The objective of this study was to derive and characterize iPSCs from karyotypically abnormal adult canine cells. Aneuploid adipose‐derived mesenchymal stromal cells (AdMSCs) from an adult female Weimeraner were re‐programmed into iPSCs via overexpression of four human pluripotency factors (Oct 4, Sox2, Klf4 and c‐myc) using retroviral vectors. The iPSCs showed similarity to human ESCs with regard to morphology, pluripotency marker expression and the ability to differentiate into derivatives of all three germ layers in vitro (endoderm, ectoderm and mesoderm). The iPSCs also demonstrated silencing of the viral transgenes and re‐activation of the silent X chromosome, suggesting full reprogramming had occurred. The levels of aneuploidy observed in the AdMSCs were maintained in the iPSCs. This finding demonstrates the potential for generating canine induced pluripotent stem cells for use as disease models in addition to regenerative medicine and pharmaceutical testing.     

Estimating the mass of asteroid 253 mathilde from tracking data during the NEAR flyby

The terminal navigation of the Near Earth Asteroid Rendezvous (NEAR) spacecraft during its close flyby of asteroid 253 Mathilde involved coordinated efforts to determine the heliocentric orbits of the spacecraft and Mathilde and then to determine the relative trajectory of the spacecraft with respect to Mathilde. The gravitational perturbation of Mathilde on the passing spacecraft was apparent in the spacecraft tracking data. As a result of the accurate targeting achieved, these data could be used to determine Mathilde's mass as 1.033 (+/- 0.044) x 10(20) grams. Coupled with a volume estimate provided by the NEAR imaging team, this mass suggests a low bulk density for Mathilde of 1.3 grams per cubic centimeter.     

The effect of different combinations of lignocaine,ketoprofen, xylazine and tolazoline on the acute cortisol response to dehorning in calves

AIMS: The aims of this study were (a) to evaluate the effect of xylazine and tolazoline, with and without lignocaine, on the cortisol response of calves following amputation dehorning and (b) to assess the effect of a non-steroidal anti-inflammatory drug (ketoprofen) and local anaesthesia on the cortisol response of calves to amputation dehorning.

METHODS: Plasma cortisol concentrations were measured in 100 dehorned or non-dehorned 3-month-old calves over an 8-h period following five different sedative/analgesic or control treatments. Sedative/analgesic treatments were: control (no anaesthesia); local anaesthesia and ketoprofen; local anaesthesia and xylazine; local anaesthesia, xylazine and tolazoline; and xylazine only. Within each sedative/analgesic treatment group, half the calves (n=10 per group) were amputation dehorned and half were not dehorned.

RESULTS: The change in plasma cortisol concentrations in calves dehorned after being given ketoprofen and local anaesthesia did not differ significantly from that of non-dehorned control calves for at least 8 h. In contrast, the cortisol response of dehorned calves not given analgesic drugs peaked 30 min after dehorning and lasted >4 h. Xylazine injected before dehorning significantly reduced but did not eliminate the peak of the cortisol response. When both xylazine and local anaesthesia were administered before dehorning the peak in the cortisol response was virtually eliminated. In the dehorned calves that received xylazine with or without local anaesthesia, cortisol concentration increased significantly 3 h after dehorning and did not return to baseline until at least 5 h later. When tolazoline was administered shortly after xylazine, it caused a marked cortisol response, higher than the response to any other treatment.

CONCLUSIONS: Combining ketoprofen and local anaesthesia minimised the cortisol response, and by inference the pain- induced distress, following amputation dehorning in calves. Xylazine reduced the initial cortisol response to dehorning but not as much as when local anaesthesia was also given. The increase in cortisol concentration from 3–8 h after dehorning in calves given xylazine alone or in combination with local anaesthesia suggests that calves experienced pain-induced distress during this time and that xylazine had no long-term analgesic effect. Tolazoline, used to reverse the sedative effects of xylazine, caused a marked cortisol response in calves via a mechanism which remains unclear.     

Sucking behaviour of hand-reared newborn dairy calves

AIM: To determine the feeding ability of calves during the first 4 days after birth.

METHODS: The sucking behaviour of 171 dairy calves, fed from individual bottles during the first 4 days after birth, was evaluated by quantifying the volume of colostrum consumed, the duration of feeding, and speed of drinking. Calves had been separated from their mothers and brought into a rearing shed in the morning, when <24 h of age, and were offered 2 L colostrum from a bottle in the afternoon of the same day, and twice daily thereafter.

RESULTS: Newborn calves became efficient suckers from bottles within 24 h of removal from their dams (collection). On the day after birth, 95% of calves drank the 2 L of colostrum offered during the afternoon feed, and by Day 4 virtually all calves (99%) drank this amount. Calves that had inadequate colostrum from their dam were more likely to drink all 2 L offered after collection, but a small number of calves that had not had colostrum from their dams and drank <500 ml at the afternoon feed following collection were likely not to drink 2 L on the following days. However, calves that did not drink all 2 L on Day 1 were not disadvantaged in comparison to those that did, in that they were equally likely to drink 2 L on Day 4.

CONCLUSIONS: The majority of calves became efficient drinkers within 48 hours of birth, but a small number of slow feeders may need individual feeding at least up to 4 days after birth.     

Long-term survival after combination chemotherapy for bilateral renal malignant lymphoma in a dog

CASE HISTORY: A 6-year-old, entire male Flat-coated Retriever was presented with a history of lethargy, polydipsia and seizures. Clinical chemistry had shown marked azotaemia.

CLINICAL FINDINGS AND DIAGNOSIS: Radiography and ultrasonography revealed bilateral renomegaly, and cytology of fine needle aspirates from the kidneys was diagnostic of malignant lymphoma. The dog was treated with a modified high-dose cyclophosphamide-, vincristine-, and prednisolone-based chemotherapy protocol, achieved remission, and returned to normal quality of life. Survival time was 346 days from the time of diagnosis.

CLINICAL RELEVANCE: Malignant lymphoma in the kidneys of dogs has been considered to carry a uniformly poor prognosis. Long-term remission after medical treatment has not previously been reported. The favourable outcome in this case illustrates the limitations of clinical staging in determining the outcome for individual patients.     

Antimicrobial Resistance in Some Gram‐Negative Bacteria Isolated from the Bovine Ejaculate

The bacterial load and degree of antibiotic resistance present in untreated and antibiotic‐treated semen samples were investigated in five bulls standing at a cattle‐breeding centre. Bacterial load was determined by colony counts from semen samples cultured on brain heart infusion and nutrient agar plates. Antibiotic resistance in these bacteria was assessed by measuring the diameter of bacterial growth inhibition zones around discs containing different concentrations of antibiotics. Representative antibiotic‐resistant bacterial isolates were selected for identification. Untreated semen contained few culturable bacteria, and all were completely sensitive to gentamycin, spectinomycin and lincomycin: six of the isolates showed some resistance to tylosin. In semen to which antibiotics had been added as part of the routine production process, two isolates were sensitive to all of the antibiotics tested, and the remainder were resistant to all. Resistant Gram‐negative isolates that were identified included Pseudomonas and Stenotrophomonas spp. both in the class Gammaproteobacteria and a Sphingomonas sp. which is in the class Alphaproteobacteria.     

Effect of N‐acetyl‐L‐cysteine Supplementation in Semen Extenders on Semen Quality and Reactive Oxygen Species of Chilled Canine Spermatozoa

The objective of this study was to evaluate the quality of chilled dog semen processed with extenders containing various concentrations of N‐acetyl‐L‐cysteine (NAC). Ejaculates from five dogs were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF‐movement), viability, acrosomal integrity and by the hypo‐osmotic swelling test (HOST). In addition, superoxide anion (O₂^‐?) production, hydroxyl radicals (OH^?) and total reactive oxygen species (tROS) were determined. The pool was divided into five aliquots, which were diluted to a final concentration of 66.66 × 10⁶ spermatozoa/ml with Tris‐glucose‐egg yolk extender containing one of the following concentrations of NAC (0, 0.5, 1, 2.5 or 5 mm ). The semen aliquots were chilled and preserved at 4°C. Semen quality was evaluated after rewarming at 72 h. Sperm motility was significantly higher with the 0.5 mm concentration compared with the control group (p = 0.001). Rapid steady forward movement was higher with the 0.5 and 1 mm concentrations compared with the control and 5 mm group (p < 0.001). Viability and HOST percentages were not significantly altered. Compared with the control, the 5 mm concentration showed significantly reduced percentages of spermatozoa with normal acrosomes (p = 0.049). None of the ROS values at 72 h were significantly affected by the presence of NAC in semen extenders, although all NAC concentrations showed lower O₂^‐? and OH^? values compared with the control. Only the concentrations of 1 and 5 mm inhibited the significant increase of tROS values after 72 h, compared with the fresh semen value. In conclusion, NAC supplementation of semen extenders is beneficial to semen motility of canine spermatozoa during chilling with the 0.5 mm concentration being the most effective, although no significant ROS inhibition was observed at 72 h.     

Clinical evaluation of protein electrophoresis of cerebral spinal fluid

Protein fractions of cerebral spinal fluid samples were analyzed in an effort to find a reliable, minimally invasive and cost effective method of diagnosing cervical vertebral myelopathy. There were six separate protein fractions analyzed by electrophoresis. In this study we specifically looked for a post beta spike in horses with spinal cord compression. There was no correlation in our study between horses with a post beta spike and CVM as diagnosed by a myelogram.     

Bragg diffraction from crystallized ion plasmas

Single crystals of a one-component plasma were observed by optical Bragg diffraction. The plasmas contained 10(5) to 10(6) single-positive beryllium-9 ions (9Be+) at particle densities of 10(8) to 10(9) per cubic centimeter. In approximately spherical plasmas, single body-centered cubic (bcc) crystals or, in some cases, two or more bcc crystals having fixed orientations with respect to each other were observed. In some oblate plasmas, a mixture of bcc and face-centered cubic ordering was seen. Knowledge of the properties of one-component plasma crystals is required for models of white dwarfs and neutron stars, which are believed to contain matter in that form.