Technical note: viability and motility of vitrified/thawed primordial germ cell isolated from common carp (Cyprinus carpio) somite embryos

The feasibility of cryopreserving common carp (Cyprinus carpio) primordial germ cells (PGC) by vitrification of whole embryos at the 22- to 28-somite stage was investigated. Green fluorescent protein (GFP)-labeled PGC were cooled rapidly using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (ethylene glycol or dimethyl sulfoxide, 30 or 50 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose (5, 10, 20, or 30 min). Embryonic cells that were pretreated for 30 min and vitrified for 20 min with ethylene glycol had the greatest rate of survival of embryonic cells (68.6%; P < 0.01), an optimal highest percentage of viable PGC (73.8 to 74.9%; P < 0.05), and no evidence of ice formation after thawing. The vitrified/thawed PGC were transplanted into blastula-stage embryos from goldfish (Carassius auratus). The PGC maintained their motility and moved to the gonadal ridge of the host embryo. Thus, the combination of vitrification and transplantation to produce germ-line chimeras is a powerful tool for the artificial production of next-generation offspring.     

Heterotopic implantation of a porcine bioprosthetic heart valve in a dog with aortic valve endocarditis

CASE DESCRIPTION: A 5-year-old male German Shepherd Dog was evaluated because of a 5-month history of progressive lethargy, weight loss, and heart failure. CLINICAL FINDINGS: On physical examination, bounding femoral pulses and systolic and diastolic murmurs were detected. Echocardiography revealed severe aortic valve insufficiency (AVI) and a large vegetative lesion on the aortic valve consistent with aortic valve endocarditis. The AVI velocity profile half-time was 130 milliseconds; the calculated peak systolic pressure gradient across the aortic valve was 64 mm Hg. Left ventricular diameter during diastole was 63.6 mm (predicted range, 40.2 to 42 mm) and during systole was 42.9 mm (predicted range, 25.4 to 27 mm). Systolic, diastolic, and mean arterial blood pressures were 120, 43, and 65 mm Hg, respectively. TREATMENT AND OUTCOME: To palliate severe AVI, the descending aorta was occluded (duration, 16.75 minutes) and heterotopic implantation of a porcine bioprosthetic heart valve in that vessel was performed. After surgery, systolic, diastolic, and mean arterial blood pressures were 115, 30, and 61 mm Hg, respectively, in the forelimb and 110, 62, and 77 mm Hg, respectively, in the hind limb. Within 6 months, the AVI velocity profile half-time had increased to 210 milliseconds, indicating diminished severity of AVI. After 24 months, the dog was able to engage in vigorous exercise; no pulmonary edema had developed since surgery. CLINICAL RELEVANCE: Heterotopic bioprosthetic heart valve implantation into the descending aorta during brief aortic occlusion appears feasible in dogs and may provide substantial palliation for dogs with severe AVI.     

Supplementing transglucosidase with a high-fiber diet for prevention of postprandial hyperglycemia in streptozotocin-induced diabetic dogs

Indigestible oligosaccharides have been shown to normalize blood glucose and insulin concentration thereby promoting good health and preventing diseases, such as diabetes. Transglucosidase (TG, α-glucosidase, enzyme code (EC) 3.2.1.20) is an enzyme capable of converting starch to oligosaccharides, such as iso-malto-oligosaccharides from maltose, via the action of amylase. The aim of this study was to evaluate whether oral administration of TG with maltose or dextrin is capable of reducing post-prandial serum glucose concentration in experimentally streptozotocin (STZ)-induced diabetic dogs fed on a high-fiber diet. Five healthy and five STZ-induced diabetic dogs were employed in this study. TG supplementation with dextrin or maltose had no detrimental effect in healthy dogs. In fact, TG and dextrin exhibited a flatlined serum glucose pattern, while reducing mean post-prandial serum insulin and glucose concentration as compared to control diet alone. When TG supplementation was tested in STZ-induced diabetic dogs under the context of a high fiber diet, a 13.8% and 23.9% reduction in mean glucose concentration for TG with maltose and dextrin, respectively was observed. Moreover, TG with dextrin resulted in a 13% lower mean post-prandial glucose concentration than TG with maltose, suggesting that dextrin may be a more efficient substrate than maltose when used at the same concentration (1 g/kg). Our results indicate that TG supplementation with diet can lead to lower postprandial glucose levels versus diet alone. However, the efficacy of TG supplementation may depend on the type of diet it is supplemented with. As such, TG administration may be useful for preventing the progression of diabetes mellitus and in its management in dogs.     

Cloning of cDNAs encoding multiple subtypes of feline interferon-alpha from the feline epitherial cell line

Mammalian interferon (IFN)-alpha consists of a 23-amino acid signal peptide and a 166-amino acid mature protein. Feline (Fe) IFN-alpha has an extra unique molecule consisting of a 171-amino acid mature protein with a 5-amino acid insertion. We cloned eight new subtypes of cDNA encoding FeIFN- alpha from a feline epithelial cell line. Among all the FeIFN-alpha subtypes, including six that have previously been reported, the variations were found to be far less than those of IFN-alphas of other animals.     

Purification of swine carbonic anhydrase isoenzyme III and measurement of its levels in tissues and plasma

The changes in the levels of carbonic anhydrase isozyme III (CA‐III) in swine plasma and urine have not been previously determined or reported. CA‐III is relatively specific to skeletal muscles, and should therefore be a useful diagnostic marker for muscle diseases. We isolated CA‐III from swine muscle tissues and determined CA‐III levels in the plasma and urine from both healthy and diseased pigs. The levels of CA‐III in the tissues of female swine (age, 3 months) and plasma of young swine (age, 1–5 months) and adult female pigs (age, 2–3 years) were determined using the ELISA system for swine CA‐III. The mean (± SD) levels of CA‐III in the skeletal muscles were 3.8 ± 3.2 mg/g (wet tissue), and in the plasma, 230 ± 193 ng/ml at 1 month, 189 ± 208 ng/ml at 2 months, 141 ± 148 ng/ml at 3 months, 78 ± 142 ng/ml at 4 months and 53 ± 99 ng/ml at 5 months. The mean level of CA‐III in the plasma samples from 2‐ to 3‐year‐old pigs was 18 ± 60 ng/ml. CA‐III in the plasma samples was found to decrease from 1 month until 3 years of age (p < 0.01). We performed far‐western blotting to clarify the cause of the observed decrease in CA‐III in plasma. Our results demonstrated that CA‐III is bound to the transferrin and albumin. In addition, we determined that the levels of CA‐III in plasma and urine samples were higher in diseased swine compared with the healthy pigs.     

Expression of desmosomal proteins in rat keratinocytes during in vitro differentiation

The keratinocyte, the major component of the epidermis, expresses several proteins that characterize the keratinization during the differentiation. Proliferation and differentiation of cultured human keratinocytes are known to be regulated by the Ca2+ concentration in the culture medium. However, informations about the rat keratinocyte are relatively limited and their physiology is still an open question. To elucidate the characteristics of the rat keratinocyte, we established rat keratinocyte culture system and examined effects of extracellular calcium concentration on the expression of differentiation-related proteins. Keratinocytes were isolated from the newborn rat skin with 0.25% trypsin, followed by separation with a Percoll density gradient. The separated cells were grown in MCDB 153 medium containing several growth factors and Ca(2+)-free fetal bovine serum, then stimulated with Ca2+. Immunoblotting demonstrated strong expression of beta1 integrin in unstimulated cells, suggesting that the primary culture of rat keratinocytes was successfully established. Expression of desmoglein and transglutaminase was increased by Ca2+ stimulation, whereas beta1 integrin expression was decreased in response to increasing concentrations of Ca2+. These observations indicate that cultured rat keratinocytes maintain the ability to differentiate in vitro, which is similar to that of the basal keratinocytes in the epidermis.     

A Survey for the Microfilariae of the Canine Heartworm, Dirofilaria immitis, in the Calgary Region of Southern Alberta

A blood survey for the prevalence of the microfilariae of the canine heartworm, Dirofilaria immitis was conducted in the Calgary area of southern Alberta between November 1977 and August 1979. A total of 514 blood samples was examined by the modified Knott's test. All of the samples were negative for D. immitis microfilariae. Wright's stained blood smears taken from 19 animals at the Calgary Zoo also proved negative. One smear from a male two-toed sloth (Choloepus didactylus) contained sheathless microfilariae which were distinguishable from those of D. immitis. These results, as well as mail survey data indicate that D. immitis is not endemic in the Calgary area.

It is recommended that the modified Knott's test be used for similar large scale sampling studies. In addition, it is suggested that the testing of both native and exotic zoo animals which could serve as definitive hosts of D. immitis be continued. These animals may become local sources of infection or introduce other species of microfilariae which will have to be differentiated from those of D. immitis.

     

Evaluation of serum and urine 1,5-anhydro-D-glucitol and myo-inositol concentrations in healthy dogs

1,5-anhydro-D-glucitol (1,5AG) is a pyranoid polyol compound found in human circulating blood. Myo-inositol (MI) is a stereoisomer of inositol and serves as a precursor of inositol phospholipids. 1,5AG and MI are filtered by the glomerulus and almost completely reabsorbed through the renal tubules. However, under hyperglycemic conditions, reabsorption through the renal tubules is competitively inhibited because the structures of 1,5AG and MI resemble that of glucose. In this study, we investigated the kinetics of serum and urine 1,5AG and MI levels in healthy dogs. We demonstrated that 1,5AG and MI exist in canine serum and urine by gas chromatography-mass spectrometry. Under continuous hyperglycemic conditions, the serum 1,5AG concentration in healthy dogs decreased while the serum MI concentration remained unchanged. Urinary excretion of 1,5AG and MI increased significantly after blood glucose concentrations reached 200 to 220 mg/dl. A significant negative correlation was observed between serum 1,5AG and glucose concentrations during hyperglycemia. However, no significant correlation was observed between serum MI and glucose concentrations. In this study, we demonstrated that serum and urine 1,5AG and MI levels were changed by blood glucose concentrations. The serum 1,5AG concentration was decreased by continuous hyperglycemia. However, the serum MI concentration does not reflect hyperglycemia.