Insulin-like growth factor binding protein-3: its biological effect on bovine granulosa cells

This study was aimed at testing the hypothesis that insulin-like growth factor binding protein (IGFBP)-3 can modulate hormone-dependent differentiation of granulosa cells in vitro. Granulosa cells from small (1 to 5 mm) follicles were collected from cattle, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 d in serum-free medium with follicle-stimulating hormone (FSH) (50 ng/ml), recombinant human IGF-I (0, 1.3, 4.0, or 13.3 nM), or recombinant human IGFBP-3 (0 to 4.26 nM). In one series of experiments, IGFBP-3 (0.53 and 2.13 nM) inhibited (51% to 92% decreases; P < 0.05) progesterone and estradiol production induced by 1.3 nM of IGF-I, but did not influence (P > 0.10) granulosa cell numbers or steroidogenesis in the absence of IGF-I. Only 4.26 nM of IGFBP-3 inhibited (by 35%) the increase in granulosa cell numbers induced by 1.3 nM of IGF-I. In another series of experiments, 13.3 nM of IGF-I, but not 4.0 nM of IGF-I, was able to completely overcome the inhibitory effect of 4.26 nM of IGFBP-3 on estradiol production. The increase in cell numbers induced by 4.0 and 13.3 nM of IGF-I was attenuated (P < 0.001) by 4.26 nM of IGFBP-3. In a third series of experiments, IGFBP-3 inhibited 125I-IGF-I binding to granulosa cells. These results indicate that IGFBP-3 has a pronounced inhibitory effect on IGF-I action in cultured bovine granulosa cells, and that this inhibitory effect is likely attributable to IGFBP-3 binding/sequestering IGF-I. Thus, IGFBP-3 may play a significant role in regulating granulosa cell proliferation and steroidogenesis during follicular development in cattle.     

Oligodendroglial vacuolar degeneration in the bilateral motor cortices and astrocytosis in epileptic beagle dogs

We performed a pathologic examination of the brains of three dogs in an epileptic beagle colony. Histologically, all the cases had diffuse astrocytosis in the cerebral cortex and basal ganglia as well as the hippocampus, whereas they showed acute nerve cell change in the hippocampus and some other areas of the cerebrum. One of these animals showed laminar myelin pallor associated with the presence of many vacuoles in the IV to VI layers of the bilateral motor cortices. Most of the vacuoles contained fine granules stained with luxol-fast-blue stain. Ultrastructural examination revealed that some oligodendrocytes and perineuronal satellite oligodendrocytes in the bilateral cerebral motor cortices of the two affected dogs had many vacuoles surrounded by myelin-like lamellar structures. These findings suggest a possibility that astrocytosis in the cerebrum and vacuolar degeneration of oligodendrocytes in the cerebral motor cortex may be, at least in part, related to the occurrence or development of seizures.     

Use of a water hardness test kit to measure serum calcium concentration in cattle

OBJECTIVE: To determine whether a commercially available water hardness test kit could be used to measure total serum calcium concentration and diagnose hypocalcemia in dairy cows. DESIGN: Prospective study. ANIMALS: 30 dairy cows from 19 commercial herds. PROCEDURE: Serum calcium concentration was determined using a water hardness test kit and a standard, laboratory-based method. Simple linear regression was used to determine whether there was a linear relationship between results of the 2 methods, and Spearman's rank correlation was used to calculate correlation between measurements. Sensitivity, specificity, and predictive values of using test kit-derived values for diagnosis of hypocalcemia (laboratory value < 8 mg/dl) were calculated. RESULTS: There was a high correlation and significant linear relationship between results of the 2 methods. Sensitivity, specificity, predictive value of a positive test result, and predictive value of a negative test result were 100, 73, 86, and 100%, respectively. Accuracy was improved by using a test kit-derived calcium concentration of 7 mg/dl as the cut-off for determining hypocalcemia. CLINICAL IMPLICATIONS: Results indicate that a commercially available water hardness test kit can be used as a rapid, inexpensive method of estimating serum calcium concentrations and diagnosing hypocalcemia in dairy cattle. However, the test is not practical for cow-side use, because blood samples must be centrifuged to obtain serum for use in the test kit.     

Effect of dietary n-6-to-n-3 fatty acid ratio on complete blood and total white blood cell counts, and T-cell subpopulations in aged dogs

OBJECTIVE: To determine effect of diets with variable n-6-to-n-3 fatty acid (FA) ratio on CD4+ and CD8+ T-lymphocyte subpopulations, and on results of routine laboratory analyses (CBC and total WBC count, serum biochemical analyses, and urinalysis). ANIMALS: 20 healthy, aged (9.5 to 11.5 years old) female Beagles. PROCEDURE: Dogs were fed 1 of 3 diets that contained 6% fat by weight but differed in amounts of n-6 and n-3 FA. For 11 weeks, 6 dogs were fed a low concentration of n-3 FA (ratio, 31:1), 7 were fed a medium concentration (5.4:1), and 7 were fed a high concentration (1.4:1). Preprandial blood and urine samples were collected before beginning the study and at 8 weeks for evaluation of laboratory variables. Before and at 3, 6, and 8 weeks during the study, blood was drawn for total WBC and lymphocyte counts and for characterization of T-cell subpopulations. At 8 and 10 weeks, dogs were vaccinated with keyhole limpet hemocyanin suspension. Blood was drawn 4 days after each vaccination, and lymphocytes were isolated for flow cytometry. Effects of diet and vaccination on each variable were determined. RESULTS: After vaccination, total lymphocyte count increased and CD4+ T lymphocyte count and the CD4(+)-to-CD8+ ratio decreased in dogs consuming the diet with n-6-to-n-3 FA ratio of 1.4:1. CONCLUSION: Feeding a diet with n-6-to-n-3 FA ratio of 1.4:1 had significant effects on CD4+ T lymphocytes in healthy, aged Beagles after vaccination.     

Seroprevalence of antibodies against gram-negative core antigens in rabbits, using an Escherichia coli J5 antigen-capture enzyme-linked immunosorbent assay

OBJECTIVE: To determine the seroprevalence of antibodies to gram-negative core antigens (GNCA) in specific-pathogen-free (SPF) rabbits (ie, free of Pasteurella multocida) and rabbits of undefined bacterial status (conventional). SAMPLE POPULATION: Serum samples were obtained from 7 groups of rabbits. The SPF rabbits comprised 2 adult groups and 1 immature group, whereas the 4 groups of conventional rabbits were all adults. PROCEDURE: A seroprevalence survey was conducted on rabbit sera for antibodies against GNCA, using an Escherichia coli J5 antigen-capture ELISA. RESULTS: Collective geometric mean titer (GMT) of adult rabbits was 1:6,463. The GMT of each of the 6 groups of adult rabbits was 1:956, 1:1,133, 1:4,525, 1:5,338, 1:7,669, and 1:25,600. Titers of populations differed significantly. CONCLUSION: Data analysis revealed there were anti-GNCA antibodies in rabbits. Similar to other species, the prevalence of IgM and IgG anti-GNCA antibodies increased with age. The IgG response was more marked than the IgM response. The SPF rabbits had lower IgG anti-GNCA titers than conventional rabbits, indicating possible cross-reactive epitopes between P multocida and Enterobacteriaceae. Rabbits with the highest anti-GNCA titers were those used in polyclonal antibody production, possibly stemming from endotoxin contamination of antigen or adjuvant. CLINICAL RELEVANCE: The possible cross-reactive antibodies directed at homologous wall components of Pasteurellaceae and Enterobacteriaceae could prove to be a possible heterotypic vaccination strategy for the protection of rabbits against pasteurellosis. Investigators should determine whether antigen impurity (endotoxin contamination) influences epitope focus during polyclonal antibody production and whether it affects sera variability among rabbits.     

Indirect ELISAs based on recombinant and affinity-purified glycoprotein E of Aujeszky's disease virus to differentiate between vaccinated and infected animals

Two indirect ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera have been developed. The rec-gE-ELISA is based on the E. coli-expressed recombinant protein containing the N-terminal sequences of gE (aa 1-125) fused with the glutathione S-transferase from Schistosoma japonicum. The affi-gE-ELISA is based on native gE, which was purified from virions by affinity chromatography. The tests were optimised and compared with each other, as well as with the recently developed blocking gE-ELISA (Morenkov et al., 1997b), with respect to specificity and sensitivity. The rec-gE-ELISA was less sensitive in detecting ADV-infected animals than the affi-gE-ELISA (sensitivity 80% and 97%, respectively), which is probably due to the lack of conformation-dependent immunodominant epitopes on the recombinant protein expressed in E. coli. The specificity of the rec-gE-ELISA and affi-gE-ELISA was rather moderate (90% and 94%, respectively) because it was necessary to set such cut-off values in the tests that provided a maximum level of sensitivity, which obviously increased the incidence of false positive reactions. Though the indirect ELISAs detect antibodies against many epitopes of gE, the blocking gE-ELISA, which detects antibodies against only one immunodominant epitope of gE, showed a better test performance (specificity 99% and sensitivity 98%). This is most probably due to rather high dilutions of the sera used in the indirect gE-ELISAs (1:30) as compared to the serum dilution in the blocking gE-ELISA (1:2). We conclude that the indirect gE-ELISAs are sufficiently specific and sensitive to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine and can be useful, in particularly affi-gE-ELISA, as additional tests for the detection of antibodies to gE.     

Analysis of correlated continuous repeated observations: modelling the effect of ketosis on milk yield in dairy cows

This study used mixed models analysis to demonstrate the advantages of a repeated measures technique for a continuous variable over a single measure technique. As an illustration, the loss of milk yield due to ketosis was studied in 2604 multiparous New York State Holstein cows belonging to eight herds, calving between 1991 and 1993. Two methods of analysis were presented: The first treated milk yield as a continuous, summary measure (projected 305-day milk yield); the second treated milk yield as repeated measurements (test-day milk yields). In the first example, with 305-day milk yield as the outcome, ketosis was treated as a binary covariate. Ketosis had no effect on the 305-day milk yield. In the second example, with monthly test-day milk yields as the outcome, four different covariance structures (simple, compound symmetry, autoregressive, and unstructured) to model the association among the repeated measurements were compared. With this approach, ketotic cows yielded significantly less milk per day both before and immediately after diagnosis than did non-ketotic cows. Based on the goodness-of-fit statistics, it was unclear whether an autoregressive or unstructured covariance structure was best. However, an autoregressive structure, in which the previous and current test-day milk yields are assumed to be correlated, was considered more suitable in this study; it is a simpler and more appropriate covariance structure for this particular problem than is an unstructured covariance structure. Nevertheless, with the test-day approach, any of these correlation structures could be used to estimate milk loss after disease. Based on these findings, it is recommended that a repeated measures approach, rather than a single measure approach, be used to study the short-term effect of disease on milk yield.     

In vitro dose-dependent effects of enrofloxacin on equine articular cartilage.

OBJECTIVE: To determine whether enrofloxacin has detrimental, dose-dependent effects on equine articular cartilage in vitro. ANIMALS: Cartilage explants were developed from 6 healthy horses between 0 and 96 months old. PROCEDURE: Patellar cartilage explants were incubated in 5 concentrations of enrofloxacin (2 microg/ml, 10 microg/ml, 1,000 microg/ml, 10,000 microg/ml, and 50,000 microg/ml) for 72 hours. Proteoglycan synthesis (Na35SO4 incorporation for 24 hours), proteoglycan degradation (Na35SO4 release for 72 hours), endogenous proteoglycan content (dimethylmethlene blue assay), and total protein content were determined. Cartilage explants were evaluated by use of histomorphologic and histomorphometric techniques (toluidine blue stain) for cytologic and matrix characteristics. Quantitative data were analyzed with a one-way ANOVA to compare results among various enrofloxacin concentration groups and the control group. A general linear model was used to determine whether age had an effect. RESULT: Proteoglycan synthesis was excellent in control specimens and in specimens incubated in low concentrations of enrofloxacin (2 microg/ml and 10 microg/ml). High concentrations of enrofloxacin (> 1,000 microg/ml) effectively eliminated proteoglycan synthesis regardless of horse age. Proteoglycan degradation at low concentrations (2 microg/ml and 10 microg/ml) was not different than control. High concentrations of enrofloxacin (> 1,000 microg/ml) caused significant degradation. Different concentrations of enrofloxacin did not affect endogenous proteoglycan. High concentrations of enrofloxacin were associated with a significant increase in number of pyknotic nuclei. CONCLUSION: Concentrations of enrofloxacin that might be achieved following systemic administration did not suppress chondrocyte metabolism in vitro. High concentrations of enrofloxacin (> 1,000 microg/ml) were toxic to chondrocytes.     

Inhibition of Salmonella typhimurium in the chicken intestinal tract by a transformed avirulent avian Escherichia coli.

An avirulent, wild-type avian Escherichia coli (E. coli Av) was electrotransformed with a plasmid coding for the production of microcin 24 (pGOB18) and was designated E. coli AvGOB18. The transformant inhibited the growth of seven serotypes of Salmonella commonly associated with colonization and contamination of poultry products and seven strains of E. coli O157:H7 in the in vitro colicin/microcin assay. The transformant did not inhibit the replication of multiple isolates of Listeria monocytogenes or Campylobacter jejuni in similar assays. The transformant is nonconjugative, indicating that the plasmid would not be transmitted to other intestinal microflora in the environment. The transformant also survived in sterile tap and deionized water incubated at 25 C and 37 C in the laboratory for 30 days and was recovered from drinkers and birds in in vivo floor pen studies. In in vivo studies, E. coli AvGOB18 did not colonize the intestinal tract of broiler chicks when given as a single or multiple dose and did not reduce the Salmonella load in the broilers. But Salmonella typhimurium was reduced significantly in the intestinal tracts of broiler chickens when E. coli AvGOB18 was administered continually in the water supply.     

Performance and digestibilities of beef cattle fed diets supplemented with either soybean meal or roasted soybeans and implanted with Synovex.

Two 160-d feedlot experiments, each consisting of 20 Angus-Hereford steers (216 +/- 5 kg BW, Exp. 1; 258 +/- 5 kg BW, Exp. 2) and 20 Angus-Hereford heifers (208 +/- 5 kg BW, Exp. 1; 236 +/- 5 kg BW, Exp. 2), were used to investigate the effects of supplementing diets with either roasted soybeans (RSB, roasted at 127 degrees C for 10 min) or soybean meal (SBM) and implanting or not implanting with an estrogenic growth promoter (SYN; Synovex-S, 20 mg of estradiol benzoate plus 200 mg of progesterone or Synovex-H, 20 mg of estradiol benzoate plus 200 mg of testosterone) on performance. The cattle were fed a basal diet of 15% orchardgrass silage, 15% corn silage, and 70% corn-based concentrate. Treatments were 1) no SYN and fed a SBM-supplemented diet, 2) no SYN and fed a RSB-supplemented diet, 3) SYN and SBM, and 4) SYN and RSB. Cattle in the SYN groups were reimplanted at 80 d. Four additional Angus-Hereford steers were used in a digestion and nitrogen balance experiment conducted during the first half of Exp. 1. For the total 160-d feedlot experiments, DMI for RSB compared with SBM was lower (P < .01; 8.5 vs 9.2 kg/d, SEM = .07) and ADG/DMI tended to be higher (P < .10; 165 vs 157 g/kg, SEM = 1.3). Final BW of steers fed RSB was similar (P > .10) to that of steers fed SBM (473 vs 478 kg, SEM = 5.6), as was ADG (1.39 vs 1.43 kg/d, SEM = .02). Dry matter intake for SYN-implanted steers was higher (P < .01) than for steers not implanted (9.2 vs 8.5 kg/d). Likewise, final BW (491 vs 460 kg) and ADG (1.49 vs 1.33 kg/d) were higher (P < .01), and ADG/DMI (166 vs 157 g/kg) tended to be higher (P < .10), for SYN-implanted steers than for steers not implanted. During the more rapid muscle growth period (0 to 80 d), DMI for RSB compared with SBM was lower (P < .01; 7.8 vs 8.6 kg/d, SEM = .07) and ADG/DMI was similar (P > .10; 181 vs 172 g/kg, SEM = 1.8). Dry matter intake for SYN-implanted steers was higher (P < .05) than for steers not implanted (8.4 vs 8.0 kg/d), as was ADG/DMI (P < .01, 182 vs 171 g/kg). During this more rapid growth period, the supplement x implant interaction for ADG was significant (P < .05; 1.35, 1.36, 1.59, and 1.44 kg/d for Treatments 1, 2, 3, and 4, respectively, SEM = .04). There were no differences in digestibilities or N balance. The results suggest that there is no improvement in performance under feedlot conditions when RSB replaces SBM in the diet of beef cattle, and, in young cattle, RSB may reduce the response expected by an estrogenic growth promoter.