The present study examined the influence of post-cleavage time of nuclear donors on the development of reconstituted embryos in bovine nuclear transfer. Blastomeres of 16-cell stage embryos derived from in vitro-maturation, fertilization and culture were used as nuclear donor source. They were treated with 10 mu-M nocodazole for 12 hr. Blastomeres that cleaved within 3 hr after the removal of nocodazole were used-for the study. Metaphase II (M-II) oocytes were used as recipient cytoplasm. IN experiment 1, donor blastomeres at 6, 11 and 15 hr after the removal of nocodazole and donor blastomeres no treated with nocodazole were transferred into ethanol-exposed and enucleated oocytes. The reconstituted embryos produced by donor blastomeres oat 6 hr after the removal of nocodazole had a significantly higher developmental rate to the blastocyst stage than those at 15 hr and the untreated groups (P<0.01). In experiment 2, blastomeres at 6 hr after the removal of nocodazole used as nuclear donors were transferred into ethanol-exposed and enucleated M-II oocytes. The reconstituted embryos with ethanol-exposed and enucleated oocytes as recipient cytoplasm had a significantly higher rate of initial-cleavage (P<0.05) and development to the blastocyst stage (P<0.01) than non ethanol-exposed and enucleated M-II oocytes. These results demonstrate that the development of reconstituted embryos was improved when cleaved donor blastomeres after the removal of nocodazole were immediately transferred (at 3-6 hr post-cleavage) into activated enucleated oocytes by exposure to ethanol.

Trypanosomiasis has been reported in dogs from Texas, Oklahoma, Louisiana, and South Carolina. We describe the first isolation and characterization of Trypanosoma cruzi from a Walker Hound pup in Virginia that also had postvaccinal distemper. The mother of the pup and 7 of its 8 siblings also were found to be infected with T cruzi, suggesting that the parasite had been transmitted transplacentally or through lactation. Parasitologic, serologic, histologic, and molecular methods were used to establish the diagnosis of T cruzi infection in these dogs. In a serologic survey of 12 dogs (including the sire of the pups) from the area in which the index case occurred, none were found to have antibodies to T cruzi. However, 2 of a further 52 dogs from different areas (to the index case), but in the same county, were sero-positive to T cruzi. These findings indicate that canine trypanosomiasis is present in an area of the United States not previously known to be enzootic.

Thirty horses were randomly assigned to 1 of 5 groups. All horses were anesthetized and subjected to ventral midline celiotomy, then the large colon was exteriorized and instrumented. Colonic arterial blood flow was reduced to 20% of baseline (BL) and was maintained for 3 hours. Colonic blood flow was then restored, and the colon was reperfused for an additional 3 hours. One of 5 drug solutions was administered via the jugular vein 30 minutes prior to colonic reperfusion: group 1, 0.9% NaCl; group 2, dimethyl sulfoxide: 1 g/kg of body weight; group 3, allopurinol: 25 mg/kg; group 4, 21-aminosteroid U-74389G: 10 mg/kg; and group 5, manganese chloride (MnCl2): 10 mg/kg. Hemodynamic variables were monitored and recorded at 30-minutes intervals. Systemic arterial, systemic venous (SV), and colonic venous (CV) blood samples were collected for measurement of blood gas tensions, oximetry, lactate concentration, PCV, and plasma total protein concentration. The eicosanoids, 6-keto prostaglandin F1alpha, prostaglandin E2, and thromboxane B2, were measured in CV blood, and endotoxin was measured in CV and SV blood. Full-thickness biopsy specimens were harvested from the left ventral colon for histologic evaluation and determination of wet weight-to-dry weight ratios (WW:DW). Data were analyzed, using two-way ANOVA for repeated measures, and statistical significance was set at P < 0.05. Heart rate, mean arterial pressure, and cardiac output increased with MnCl2 infusion; heart rate and cardiac output remained increased throughout the study, but mean arterial pressure returned to BL values within 30 minutes after completion of MnCl2 infusion. Other drug-induced changes were not significant. There were significant increases in mean pulmonary artery and mean right atrial pressures at 2 and 2.5 hours in horses of all groups, but other changes across time or differences among groups were not observed. Mean pulmonary artery pressure remained increased through 6 hours in all groups, but mean right atrial pressure had returned to BL values at 3 hours. Mean colonic arterial pressure was significantly decreased at 30 minutes of ischemia and remained decreased through 6 hours; however, by 3.25 hours it was significantly higher than the value at 3 hours of ischemia. Colonic arterial resistance decreased during ischemia and remained decreased throughout reperfusion in all groups; there were no differences among groups for colonic arterial resistance. Colonic venous PO2, oxygen content, and pH decreased, and PCO2 and lactate concentration increased during ischemia but returned to BL values during reperfusion. Compared with BL values, colonic oxygen extraction ratio was increased from 0.5 to 3 hours. By 15 minutes of reperfusion, colonic oxygen extraction ratio had decreased from the BL value in all groups and either remained decreased or returned to values not different from BL through 6 hours. Colonic venous 6-keto prostaglandin F1alpha and prostaglandin E2 concentrations increased during ischemia, but returned to BL on reperfusion; there were no changes in thromboxane2 concentration among or within groups. Endotoxin was not detected in CV or SV blood after ischemia or reperfusion. There were no differences among or within groups for these variables. Low-flow ischemia and reperfusion (I-R) of the large colon caused mucosal injury, as evidenced by increases in percentage of surface mucosal disruption, percentage depth of mucosal loss, mucosal hemorrhage, mucosal edema, mucosal interstitial-to-crypt ratio, mucosal neutrophil index, submucosal venular neutrophil numbers, and mucosal cellular debris index. There was a trend (P = 0.06) toward greater percentage depth of mucosal loss at 6 hours in horses treated with dimethyl sulfoxide, compared with the vehicle control solution. There were no differences in the remainder of the histologic variables among groups. Full-thickness and mucosal WW:DW increased with colonic I-R, but there were no differences among groups. There was a trend (P = 0.09) toward neutrophil accumulation, as measured by myeloperoxidase activity, in the lungs after colonic I-R, but there were no differences among groups. There was no change in lung WW:DW after colonic I-R. There were no beneficial effects of drugs directed against oxygen-derived free radical-mediated damage on colonic mucosal injury associated with low-flow I-R. Deleterious drug-induced hemodynamic effects were not observed in this study.

In Zimbabwe, ocular squamous cell carcinoma (OSCC) was frequently observed in 5 breeding herds of Simmental cattle, a Bos taurus breed originating from Switzerland. In these herds, initial signs of OSCC were already noticeable in cattle about 3 years old. Gradually, OSCC prevalence increased, and 36 to 53% of cattle over 7 years old had 1 or more tumors. More tumors developed in Simmental cattle with periorbital white skin than in cattle with periorbital pigmented skin. Other breeds of cattle (eg, Friesian) also are partly white-faced and live in Zimbabwe in a comparable environment; yet, OSCC prevalence was lower in those breeds.

A series of studies was conducted to identify a practical measure for preventing dental calculus formation in dogs. The studies involved a colony of 27 Beagles that received an initial dental prophylaxis. The dogs were then stratified on the basis of their normal rate of calculus formation and randomly assigned to parallel groups within each strata. During 4-week test periods, a variety of experimental regimens were instituted, followed by clinical assessments of calculus. Major observations were that a crystal growth inhibitor, soluble pyrophosphate, incorporated into a dry dog food modestly reduced calculus formation when used at high concentrations; anticalculus effects attributable to this agent were significant (P < 0.05) only when it was used as a surface coating the coating of dry dog chow or plain biscuits with a calcium sequestrant, sodium hexametaphosphate (HMP), provided the greatest benefit and resulted in significant (P < 0.05) reductions in calculus formation of about 60 to 80%, depending on the dosage regimen; and the feeding of a single daily snack of 2 HMP-coated plain biscuits (0.6% HMP) decreased calculus formation by nearly 80%. We concluded that the coating of dry dog chow or plain dog biscuits with HMP is an effective means of reducing calculus formation in dogs.

Loline and ergot alkaloids found in endophyte-infected (Acremonium coenophialum) tall fescue (EITF) cause vasoconstriction of equine vessels in vitro. An aggregate risk study was used to evaluate the association between horses exposed to EITF and development of laminitis. Veterinary teaching hospitals participating in the Veterinary Medical Data Base were grouped by whether equine accessions were likely to have been at high, moderate, or low risk for exposure to EITF. From 1980-1990, there were 185,781 accessions, of which 5,536 had diagnosis of laminitis. Proportion of equine accessions with laminitis reported by veterinary teaching hospitals for high, moderate, and low risks, were 3.41, 3.04, and 2.00 cases/100 accessions, respectively (P < 0.0001). Comparison of the proportion of accessions with laminitis in the high- and moderate-risk groups with that in the low-risk group revealed significant differences between risk groups over all months (P = 0.063) and differences from month to month within risk groups (P = 0.0001). If the difference among risk groups is attributed entirely to exposure to EITF, the population-attributable risk is 7 cases/1,000 admissions, or 15% of all admissions for laminitis at veterinary teaching hospitals in our data base. Preliminary data support an association between hoses exposed to EITF and increased risk of laminitis; however, studies at the individual animal level are indicated to confirm this hypothesis.

We evaluated the single-cycle structural properties for axial compression, torsion, and 4-point bending with a central load applied to the caudal or lateral surface of a diaphyseal segment from the normal adult equine humerus, radius, third metacarpal bone, femur, tibia, and third metatarsal bone. Stiffness values were determined from load-deformation curves for each bone and test mode. Compressive stiffness ranged from a low of 2,690 N/mm for the humerus to a high of 5,670 N/mm for the femur. Torsional stiffness ranged from 558 N.m/rad for the third metacarpal bone to 2,080 N.m/rad for the femur. Nondestructive 4-point bending stiffness ranged from 3,540 N.m/rad for the radius to 11,500 N.m/rad for the third metatarsal bone. For the humerus, radius, and tibia, there was no significant difference in stiffness between having the central load applied to the caudal or lateral surface. For the third metacarpal and metatarsal bones, stiffness was significantly (P < 0.05) greater with the central load applied to the lateral surface than the palmar or plantar surface. For the femur, bones were significantly (P < 0.05) stiffer with the central load applied to the caudal surface than the lateral surface. Four-point bending to failure load-deformation curves had a bilinear pattern in some instances, consisting of a linear region at lower bending moments that corresponded to stiffness values from the nondestructive tests and a second linear region at higher bending moments that had greater stiffness values. Stiffness values from the second linear region ranged from 4,420 N.m/rad for the humerus to 13,000 N.m/rad for the third metatarsal bone. Differences in stiffness between nondestructive tests and the second linear region of destructive tests were significant (P < 0.05) for the radius, third metacarpal bone, and third metatarsal bone. Difference between stiffness values of paired left and right bones was not detected for any test. Four-point bending ultimate failure bending moments ranged from 260 N.m for the femur to 940 N.m for the third metatarsal bone. There was no difference in failure bending moment between the directions of applied central load for a given bone.

Because caffeine is metabolized by the hepatic P-450 cytochrome oxidase system, clearance of caffeine is an excellent quantitative test of hepatic function in human beings. It is currently used in much the same way that creatinine clearance is used to assess renal function. Caffeine clearance was measured in lactating dairy cows initially to determine the suitability of caffeine clearance as an indicator of hepatic function in cattle. Pharmacokinetic variables of caffeine were studied in 6 adult lactating dairy cows after IV administration of a single dose of caffeine sodium benzoate (2 mg of caffeine/kg of body weight). Caffeine concentration was analyzed by use of an automated enzyme immunoassay. The lower limit of detection of the assay for caffeine in serum was 0.079 micrograms/ml. Serum caffeine concentration-time curves best fit an open two-compartment pharmacokinetic model. Harmonic mean elimination half-life was 3.8 (range, 2.6 to 6.9) hours, and total clearance was 0.118 (range, 0.090 to 0.197) L/kg/h. Milk caffeine concentration was similar to serum concentration 1.5 to 24 hours after caffeine administration. Adverse effects were not observed in cows given caffeine.

A monoclonal antibody (MAB) against equine tumor necrosis factor-alpha (Eq TNF) was used to investigate the role of TNF in cytokine, eicosanoid, and metabolic responses of Miniature Horses given endotoxin. Plasma concentrations of interleukin 6 (IL-6), lactate, thromboxane A2 metabolite, and prostacyclin metabolite (6-keto-PGF(1 alpha)) were measured in 10 Miniature Horses given 0.25 micrograms of lipopolysaccharide (LPS; Escherichia coli O55:B5)/kg of body weight. Five horses were given Eq TNF MAB and 5 were given isotype-matched MAB as control. All horses were given 1.86 mg of antibody/kg by IV infusion, 5 minutes before LPS was given IV. Blood samples were taken 20 minutes before and at multiple intervals for 24 hours after LPS was given. Interleukin 6 bioactivity in plasma was measured, using IL-6-dependent cell line (B9). Eicosanoid activities were assessed by enzyme immunoassay, and plasma lactate concentration was determined enzymatically. Data were analyzed by ANOVA and Tukey's honest significant difference test for significant (P < 0.05) effect of treatment. Horses given Eq TNF MAB had significantly (P < 0.050) lower peak mean +/- SEM IL-6 (59 +/- 29 U/ml), lactate (16 +/- 2.00 mg/dl), and 6-keto-PGF(1 alpha) (254 +/- 79 pg/ml) values then did horses given control MAB (880 +/- 375 U/ml for IL-6; 26 +/- 0.04 mg/dl for lactate; and 985 +/- 290 pg/ml for 6-keto-PGF(1 alpha)). There was no effect of anti-TNF treatment on LPS-induced thromboxane A2 metabolite production. Tumor necrosis factor mediated IL-6, lactate, and prostacyclin responses, without affecting thromboxane production in horses given LPS.

Use of a combination of an effective gE gene-deleted pseudorabies virus (PRV) vaccine with a companion diagnostic kit for PRV glycoprotein gE has proven successful in several pseudorabies-eradication programs. To produce a large quantity of functional gE protein for development of a PRV-gE diagnostic kit, an Escherichia coli expression system containing the distal region of the PRV-gE gene of a PRV strain CF was constructed. The expressed protein contained 134 amino acids of gE protein (amino acids 77-210) fused to a 19-amino acids tag containing 6 histidine residues. After induction, a truncated PRV-gE polypeptide of 18-kd was expressed to about 20% of the total E coli proteins. Results of immunoblot analysis indicated that this E coli-produced PRV-gE protein reacted specifically with serum from PRv-hyperimmunized pigs and from field PRv-infected pigs, but not with serum samples from specific-pathogen-free pigs or pigs inoculated with gE-deleted PRV vaccine. These data indicate that, although the recombinant gE protein is produced in E coli, it still retains the antigenicity of the viral gE glycoprotein. Comparison between the recombinant gE protein, using immunoblot analysis with a commercial gE ELISA containing natural PRV-gE protein, revealed comparable test performance. This finding indicated that recombinant gE protein produced by E coli can be used for development of a companion serologic assay for a PRV-gE gene-deleted vaccine.