Response of plasma and tissue D-alpha-tocopherol in sheep to graded dietary levels of dl-alpha-tocopheryl acetate

The objective of this experiment was to establish the validity of using plasma alpha-tocopherol values as an index of alpha-tocopherol status in sheep. alpha-tocopherol values were obtained for tissues and blood of 40 sheep given four different dietary intakes of dl-alpha-tocopheryl acetate. Group 1 sheep were given a basal diet containing 25 to 32 mg dl-alpha-tocopheryl acetate kg-1 while groups 2, 3 and 4, comprising 10 sheep each, received the basal diet plus 200, 400 and 600 mg dl-alpha-tocopheryl acetate per sheep, respectively. Blood samples were obtained at zero time and then twice weekly for eight weeks, at which time sheep were killed and organs retrieved for tissue alpha-tocopherol analysis. Tocopherol concentrations were higher in all tissues (P less than 0.001) of sheep fed the vitamin E supplemented diets than the basal diet. Vitamin E stored in the liver of sheep at the end of the experiment (eight weeks) showed a linear response to the level of vitamin E in the diet. Blood plasma vitamin E concentrations increased following vitamin E supplementation, but no direct relationship was found between vitamin E intake and plasma vitamin E content.     

Changes in gas composition and acid-base values of venous blood samples stored under different conditions in 4 domestic species

BACKGROUND: The effect of storage temperature and time on blood gas and acid-base values has been investigated intensively in cattle and dogs; however, data are lacking in other species. OBJECTIVE: The aim of our study was to evaluate changes in gas composition and acid-base values in venous blood stored at different temperatures and for different times in 4 domestic species in Italy. METHODS: Blood samples from Comisana sheep (n = 10), Maltese goats (n = 10), Ragusana donkeys (n = 10), and Thoroughbred horses (n = 10) were analyzed after storage at 23 degrees C (room temperature) for 15 minutes (group I), 23 degrees C for 1 hour (group II), 37 degrees C for 8 hours (group III), and 4 degrees C for 24 hours (group IV). Results were analyzed using a 1-way repeated measures ANOVA. RESULTS: In all species no statistically significant differences in pH values were present in samples stored at 4 degrees C for 24 hours. This also was true for PCO2 in all species except the horse. Except for HCO3- concentration in the horse, significant changes in PO2, HCO3- concentration, base excess, and the standard bicarbonate concentration were observed for all species in samples stored at 4 degrees C. In samples stored for only 1 hour at room temperature, significant changes in most analytes were detected. CONCLUSIONS: The results of this study underline the need for rapid assessment of acid-base samples, because any delay, even for 1 hour, may affect the results.     

Changes in Plasma Progesterone Levels During Storage of Heparinized Whole Blood from Cow,Horse, Dog and Pig

Progesterone concentrations in heparinized plasma harvested immediately after blood collection were compared with levels obtained after storage of the corresponding whole blood for 2 h, 4 h, 6 h, 1 day, 2 days and 5 days at room temperature and in a refrigerator. The blood was taken during the luteal phase from 4 dogs, 4 horses, 4 pigs and 8 cows. For 4 cows the storage time was extended to 9 and 20 days. No significant effect of whole blood storage time on plasma progesterone concentrations could be shown for dogs or pigs. For the horse a slight but significant decrease was demonstrated when the blood was kept at room temperature. For the cow, however, a dramatic decrease was observed even when the blood was stored in the refrigerator. Following incubation of cow’s blood at room temperature, progesterone levels were close to zero after 1–2 days. By further extending the storage period, a reappearance of assayable progesterone could be elicited. For all species it was found that the storage of plasma at room temperature for 5–9 days did not change the progesterone concentrations.     

The persistence of foot-and-mouth disease virus on wool

SUMMARY Five Suffolk sheep, held in a high-security isolation room, were exposed for 2 hours to the aerosol of 3 mature pigs that had been infected with foot-and-mouth disease virus (FMDV), strain O₁-BFS. The fleeces of 3 of the sheep were contaminated with FMDV at 2 days post exposure (dpe), while at 5 dpe the fleeces of all 5 sheep were more extensively, and more heavily, contaminated. The persistence of FMDV on contaminated wool was examined in vitro using multiple 0.5 g samples of Merino wool that were each contaminated with one of 3 strains of FMDV in tissue-culture medium: O₁-BFS, O-Morocco (O-MOR 9/91) or an Asia 1 strain (TAI 1/90). Wool samples were held at either 4°C, 18°C or 37°C, and decay curves were established for each virus at each temperature. These curves predicted that O₁-BFS, O-MOR 9/91 and TAI 1/90 would fall below detect-able levels at 72, 70 and 48 days post contamination (pc), respectively, for wool stored at 4°C; at 11, 12 and 12 days pc, respectively, for wool stored at 18°C; and at 57, 68 and 33 hours pc, respectively, for wool stored at 37°C. For wool contaminated with O₁-BFS-infected sheep faeces, urine or blood, or with O₁-BFS-infected cattle saliva, decay curves predicted virus to persist for 5 to 11 days pc at 18°C. We demonstrated that the simulated scouring of FMDV-contaminated wool at 60° to 70°C would usually reduce virus to below detectable levels. The detergent component of the scouring process had little, if any, antiviral activity, and scouring at 20°C or 50°C had limited impact on FMDV titres . We recommend that either (1) simple storage of FMDV-contaminated wool for 4 weeks at temperatures of 18°C or higher, or (2) scouring of contaminated wool at 60° to 70°C would be sufficient to remove the threat of FMDV-contaminated wool being infectious to other animals .     

Determination of plasma vitamin C concentration in fattening cattle

Plasma vitamin C (ascorbic acid + dehydroascorbic acid) concentration is a good index of the nutritional status of vitamin C. However, the methodologies for storage and analyses have not been investigated in bovine plasma. The validity of an analytical method for bovine plasma using high performance liquid chromatography (HPLC) with a spectrophotometric detector was examined. Exogenous dehydroascorbic acid was almost completely converted to ascorbic acid during the preparation for analysis with a reducing reagent, dithioerythritol. The analytical recoveries of ascorbic acid were high. Ascorbic acid was not detected after treatment with ascorbic acid oxidase. Thus, the specificity of this method is considered to be high. Although vitamin C was stable in plasma treated by dithioerythritol at ?20°C for 6 days, vitamin C in untreated plasma significantly decreased during 3‐day storage at ?20°C. These results indicate that the HPLC method is suitable for the determination of plasma vitamin C in cattle and that the storage conditions are important for determination of plasma vitamin C. Plasma vitamin C concentration ranged between 1.49 mg/L and 3.33 mg/L in fattening cattle. This result suggests that fattening cattle show large individual variation in plasma vitamin C concentration.     

Comparative utilization of warm- and cool-season forages by cattle, sheep and goats

The quality of different classes of forage hay (C3, C4 grasses and legumes) was determined in intake and digestibility trials with mature cattle, sheep and goats. For all nine hays, DM and NDF digestibility by cattle and goats was higher (P less than .05) than by sheep, with no differences due to forage class. Cattle had a higher (P less than .01) DM intake than sheep or goats averaged across forage (92.6 vs 65.8 and 68.6 g/kg BW.75); hay intake was highest on legume, with no difference between C3 and C4 grasses. Mean NDF intake by cattle was greater than by sheep or goats (58.7 vs 39.6 and 42.6 g/kg BW.75); NDF intake for all animal species decreased in the order C4 grass greater than C3 grass greater than legume. Particle passage rates did not differ (P greater than .05) with forage class but were higher (P less than .02) for sheep and goats than for cattle. Prefeeding ruminal DM fill values, determined by emptying, were 10.6, 15.0 and 19.9 g/kg BW1.0 for alfalfa, orchardgrass and switchgrass hays fed to cattle, and 11.2, 11.3 and 16.5 g/kg BW1.0 for the same hays fed to sheep. Estimated turnover times for DM and NDF were shorter (P less than .05) for sheep than for cattle; DM turnover was longer for switchgrass than for alfalfa and orchardgrass, with no forage differences in NDF turnover between these two animal species. Results show that goats were superior to sheep in NDF digestion.     

New techniques to measure blood cholinesterase activity in domesticated animals.

A macromethod and a semimicromethod were developed to measure erythrocyte acetylcholinesterase activity in cattle, sheep, goats, horses, dogs, and swine, and to measure plasma cholinesterase activity in horses, dogs, and swine. Comparison of the 2 methods with erythrocytes of sheep, cattle, goats, and horses indicated both methods gave similar results. They can be done in a shorter time and are more sensitive than Michel's method. Normal deltapH values per minutes, with standard deviations for blood cholinesterase activity of animals of different ages, sexes, breeds, and species, were: 0.76 +/- 0.12/30; 0.65 +/- 0.10/15; 0.69 +/- 0.19/45; 0.78 +/- 0.11/45; 0.63 +/- 0.11/45; and 0.71 +/- 0.06/25 for sheep, cattle, goats, horses, dogs, and swine erythrocyte acetylcholinesterase, respectively; and 0.66 +/- 0.18/20; 0.67 +/- 0.20/30, and 0.46 +/- 0.05/60 for horses, dogs, and swine plasma cholinesterase, respectively. It was shown that either the chloride or the iodide salt of acetylcholine can be used as the enzyme substrate. tin blood samples stored at 5 C for 24 hours, there was no significant change of the enzymatic activity.     

Immunocytochemical Localization of Aromatase in The Ovary of Superovulated Cattle,Pigs and Sheep

Laurincik, J., L. Kolodzieyski, V. Elias, P. Hyttel, Y. Osawa, A. Sirotkin: Immunocytochemical localization of aromatase in the ovary of superovulated cattle, pigs and sheep. Acta vet. scand. 1994, 35, 185-191.–The localization of aromatase, an enzyme converting androgens to estrogen, in the ovaries of superovulated cattle, pigs and sheep was studied immunocytochemically in the preovulatory and postovulatory period using anti-human placental aromatase cytochrome P-450 antiserum. Immunostaining for aromatase was detected in the granulosa cells of preovulatory follicles of all species studied. Theca interna cells were stained in preovulatory follicles in the pig but not in cattle and sheep. Interstitial gland cells, cumulus cells and oocytes were unstained in all species. In cattle and pig the corpora lutea were unstained whereas they displayed staining in the sheep. Preantral and small antral follicles were unstained during both the preovulatory and postovulatory period in all species.It is concluded that granulosa cells of preovulatory follicles are the main residence for aromatase activity in superovulated cattle, pig and sheep, whereas the activity of theca interna and corpora lutea is species specific.     

Stability of canine factor VIII activity and von Willebrand factor antigen concentration in vitro

The in vitro stability of canine factor VIII activity, von Willebrand factor antigen concentration and the ratio of these two factors was studied. Samples were stored for up to 48 hours, either as plasma or as whole blood, at 4 degrees, 20 degrees and 37 degrees C. Factor VIII activity was generally stable in both plasma and whole blood samples for up to 48 hours at 4 degrees or 20 degrees C. The concentration of von Willebrand factor antigen was more stable in samples stored as plasma than whole blood, and for a shorter time than factor VIII activity. Consequently, the stability of the ratio of these two factors was relatively poor in vitro.     

Storage stability of some bovine plasma enzymes

Blood samples taken from domestic or wild ruminant animals typically require transportation to an analytical laboratory. Depending on circumstances, several hours or even a few days may pass between sampling and analysis. Several diagnostic plasma enzymes were measured in bovine blood samples immediately after sampling and after storage under a variety of conditions. Conditions studied included storing whole heparinized blood at 20 C for 6 hours, storage at 4 C for 3 and 5 days, and freezing freshly prepared plasma once and 4 times before analysis. For studies of erythrocyte enzymes, fresh erythrocytes were compared with erythrocytes frozen once, frozen 4 times, and prepared from whole blood stored for one week at 4 C. None of these conditions deteriorated erythrocyte acetylcholinesterase. The serum pseudoacetylcholinesterase and lactate dehydrogenase were not affected by any storage condition used. By contrast, acid phosphatase was significantly decreased by all storage conditions used. Ornithine carbamoyltransferase, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase were stable under some of the storage conditions tested.     

Evaluation of the effects of controlled autolysis on the immunodetection of PrP(Sc) by immunoblotting and immunohistochemistry from natural cases of scrapie and BSE

Seventeen clinically suspect scrapie sheep, and twelve suspected BSE-affected cattle were confirmed using routine histopathological examination by the detection of characteristic spongiform change in the medulla brain region taken at the level of the obex. Three sheep and four cows acquired as controls showed no spongiform change. Five aliquots of brain tissue from each of four brain regions were taken (cerebellum, medulla, frontal cerebral cortex and occipital cerebral cortex) from each of the 36 animals. One aliquot was frozen at -70 degrees C, the others were subjected to one of four autolysis regimes at 3 or 7 days at 25 degrees C or 37 degrees C. All samples were tested by Western immunoblotting for detection of PrP(Sc) using the Prionics - Check test (Prionics AG, Zurich, Switzerland). Further samples of medulla from 15 suspect scrapie cases, 10 healthy sheep, 13 suspect BSE cows and 5 healthy cows, were taken adjacent to the obex, and subjected to autolysis at 37 degrees C for 6, 12, 24 and 48 hours before being fixed in 10 per cent formal saline and subsequently examined by a routine immunohistochemical technique for detection of PrP(Sc) protein. The abnormal protein could not be detected in any of the control animals by either technique. PrP(Sc) could be detected by Western immunoblotting in at least one brain area from all the positive animals after autolysis for 7 days at 37 degrees C. The protein could be detected by immunohistochemistry in all cases which were positive by histopathological examination using all autolysis conditions. From the results of this study it is concluded that autolysis does not significantly compromise the diagnosis of scrapie or BSE by either of these diagnostic methods.     

Effects of blood processing techniques on sodium and potassium values: a comparison between Aldabra tortoises (Geochelone gigantea) and Burmese mountain tortoises (Manouria emys)

Background: Hematologic and plasma biochemical evaluations are routinely used in evaluating the chelonian patient, but appropriate processing techniques have been minimally defined.
Objectives: This study was designed to compare the effects of temperature, time, anticoagulant, and species on sodium and potassium values in the Aldabra tortoise ( Geochelone gigantea ) and the Burmese mountain tortoise ( Manouria emys ).
Methods: Blood samples from 7 Aldabra tortoises and 8 Burmese mountain tortoises were collected into tubes without anticoagulant and tubes containing lithium heparin. Sodium and potassium concentrations were measured by flame photometry in serum and plasma harvested immediately after collection and from aliquots of whole blood stored at 4°C and 25°C for 5 to 120 minutes.
Results: In Aldabra tortoises, storage time and temperature had no significant effect on potassium concentrations in heparinized blood and in blood without anticoagulant. However, sodium concentrations in serum and plasma decreased significantly in samples without anticoagulant stored at 4°C and 25°C and in heparinized samples stored at 4°C. In Burmese mountain tortoises, potassium concentrations in serum and plasma increased significantly with time in samples without anticoagulant and in heparinized samples stored at 4°C and 25°C, but the increases were less at 4°C. Sodium concentrations in serum and plasma decreased significantly in blood without anticoagulant and heparinized blood stored at 4°C and 25°C.
Conclusions: Storage of blood samples with and without anticoagulant at 4°C significantly improved the stability of potassium and sodium concentrations in both species of tortoises. Early separation of red cells from serum or plasma after blood collection is especially important to ensure the reliability of potassium measurements.     

Biochemical and functional properties of a leucocidin produced by several strains of Fusobacterium necrophorum

A soluble exotoxin (a leucocidin) which was lethal to peripheral blood leucocytes from cattle, sheep, rabbits and man (in order of decreasing sensitivity) was elaborated by a variety of isolates of Fusobacterium necrophorum when the majority of organisms were present as filaments in liquid culture. Maximum production of the leucocidin was achieved by concentrations of bacteria equivalent to between 4 times 10' and 4 times 10^B short cells per ml of culture above which no further increase in titre was observed. The ability of different batches of medium to support production of leucocidin was reflected in their capacity to enable F. necrophorum to grow to this range of concentration. Prolonged culture of the organism, resulting in a decline to below 6 in the pH of the medium was associated with a depression in the titre of leucocidin, presumably due to Its inactiviation under these conditions.
The leucocidin was stable at 4°C for at least 30 days, to extremes of pH (4 to 9) for 1 h at room temperature, and showed maximum activity in assays conducted at 37°c at pH 7 to 8. The exotoxin was inactivated by heating at 56°C for 30 min and possessed a molecular weight around 250,000 to 300,000 as determined by gel filtration and membrane partition chromatography.     

Effect of storage on bovine erythrocyte cholinesterase activity.

Bovine whole blood and washed erythrocyte samples with either sodium heparin or EDTA as the anticoagulant were stored at -24, 2 to 3, 21, or 37 C for up to 8 weeks to determine the stability of erythrocyte cholinesterase (ChE) activity. An automated colorimetric method was used to measure ChE activity. Regression analysis was used to estimate the rate of decline of ChE activity of samples to 95% of normal. Samples stored at -24 and 2 to 3 C were very stable, and ChE activity was maintained for several weeks or longer. Samples stored at 21 C maintained an activity level not less than 95% of normal for at least 9 days and those stored at 37 C for at least 2 days. The type of anticoagulant used did not appear to affect significantly the stability of activity.     

Interrelationships among liposoluble vitamins in ruminants

Plasma concentrations of vitamins A and E were examined in sheep, and a transitory decrease was observed after a single massive dose of vitamin D3 (5 X 10(6) IU) was administered orally or parenterally. Administration of a large dose of vitamin E to sheep decreased plasma retinol concentrations within 72 hours, but thereafter, the plasma retinol concentrations returned to near baseline values. Oral administration of a single pharmacologic dose of dl-alpha-tocopherol (5 g) to sheep caused a slow increase of this vitamin in the blood plasma. In cattle, a single IM administration of 3 liposoluble vitamins (A, D3, and E) at acceptable concentrations had no detectable influence on plasma alpha-tocopherol concentrations with the sampling intervals used. Plasma concentrations of alpha-tocopherol in these cattle showed a marked seasonal pattern; the concentrations increased from January to a peak in July, with a subsequent decrease in the fall. Also reported are estimates of inter- and intraindividual variation in plasma liposoluble vitamin concentrations.     

In vitro stimulation of bovine peripheral blood lymphocytes: effect of short-term storage of blood prior to lymphocyte culture

Storage of peripheral blood from Mycobacterium bovis-sensitized cattle from 1 to 48 hours at 4, 22, and 37 C was shown not to alter markedly the lymphocyte blastogenic response to M bovis-purified protein derivative. Concanavalin A-induced lymphocyte mitogenic responses were unaffected by storage of blood for 1, 24, or 48 hours at 22 C and 37 C; however, storage of blood for 48 hours at 4 C significantly lowered (P less than 0.05) mitogenic responses to concanavalin A, as compared with responses to blood kept at 22 C. Mononuclear cell recovery from stored blood at all temperatures was markedly less than that from freshly drawn blood samples. Cell recoveries were most affected by storage of blood at 4 C and 37 C.     

Stability of canine factor VIII: coagulant activity in vitro.

A pilot study was undertaken to assess the stability of canine factor VIII:coagulant (FVIII:C) activity over three days, under various storage conditions (plasma at 4, 20 and 37 degrees C, whole blood at 4 and 20 degrees C). Blood collected from normal and hemophiliac dogs was used. Both plasma and whole blood samples appeared to be stable for up to 48 h at 4 and 20 degrees C. A subsequent study evaluated FVIII:C stability at 4 and 20 degrees C when stored as whole blood only. Samples were tested at 0, 24 and 48 h after collection. At 4 degree C there was a significant decline at 24 h (p less than 0.05), from 110% to 97% (mean values). Although the mean value was further decreased at 48 h (89%) this was not significant (p greater than 0.05). No significant change in FVIII:C activity was observed in whole blood stored at 20 degrees C for 24 or 48 h (110% and 107% respectively). These results suggest that canine whole blood samples collected into sodium citrate stored at 20 degrees C are adequate for routine FVIII:C assay for up to 48 h after collection.     

Humoral and cellular immune responses in cattle and sheep inoculated with Sarcocystis

Cattle inoculated with Sarcocystis bovicanis (= Sarcocystis cruzi) and sheep inoculated with Sarcocystis ovicanis were monitored for the appearance of Sarcocystis-specific antibodies and lymphocytes in the peripheral circulation. Anti-Sarcocystis antibody was identified by enzyme-linked immunosorbent assay, whereas antigen-reactive lymphocytes were discerned by an in vitro lymphocyte blastogenic assay. The antigens used were the soluble fraction recovered from disrupted bradyzoites of mature sarcocysts. Cattle developed anti-Sarcocystis immunoglobulin (Ig)M responses, beginning 3 to 4 weeks after inoculation, and IgG1 antibody responses, beginning 5 to 6 weeks after inoculation. The increase in IgM antibody was relatively brief, returning to near preinfection levels in 2 to 3 months. In contrast, IgG1 antibody levels remained high for at least 5 to 6 months. Neither IgG2 nor IgA antibody responses were demonstrable in cattle. In sheep, the IgG antibody levels followed a time course similar to that seen in cattle, except that the increase was slightly delayed (6 to 8 weeks after inoculation was done). Measurable IgM antibody response was not seen in sheep. Cellular immunoresponsiveness as judged by in vitro lymphocyte blastogenesis in cattle was different from that in sheep. Sarcocystis-specific lymphocytes were demonstrable in the circulation of cattle within 15 days after they were inoculated, but the activity decreased rapidly. In sheep, reactive cells were not evident until 3 to 4 weeks after inoculation were done, but peripheral blood lymphocytes taken from these sheep as long as 5 to 6 weeks after the inoculations remained capable of mounting strong blastogenic responses. Neither the enzyme-linked immunosorbent assay nor the blastogenic assay showed species specificity. Animals immunized with a given species of Sarcocystis gave similar in vitro responses to antigens from the immunizing species and to other species of Sarcocystis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of selenium levels and glutathione peroxidase activity in bovine whole blood.

Blood glutathione peroxidase activity and selenium levels were found to correlate well, indicating that glutathione peroxidase activity can be used to assess blood selenium levels in beef cattle. The glutathione peroxidase activity of blood is less stable than is the selenium concentration but when blood was stored at 4 degrees C, the glutathione peroxidase activity remained constant for seven days.     

Role of insects in the transmission of bovine leukosis virus: potential for transmission by stable flies, horn flies, and tabanids

The ability of stable flies (Stomoxys calcitrans), horn flies (Haematobia irritans), and tabanids (Diptera: Tabanidae) to transmit bovine leukosis virus (BLV) was investigated. Stable flies and horn flies were fed on blood collected from an infected cow, and the flies' mouthparts were immediately removed, placed in RPMI-1640 medium, ground, and inoculated into sheep and calves. Infection of sheep occurred with mouthparts from as few as 25 stable flies or 25 horn flies. However, sheep were not infected when removal of stable fly mouthparts was delayed greater than or equal to 1 hour after blood feeding. Infection of calves occurred after inoculation of mouthparts removed immediately after feeding from as few as 50 stable flies or 100 horn flies. Infected blood, applied by capillary action to the mouthparts (labella) of 15 deer flies (Chrysops sp) and a single horse fly (Tabanus atratus) caused infection in each of 2 sheep. Infection did not occur in 2 calves inoculated daily for 5 days with mouthparts from 50 horn flies collected after feeding on a BLV-infected steer. Four calves receiving bites from 75 stable flies interrupted from feeding on a BLV-positive cow also were not infected. Seronegative cattle held for 1 to 4 months in a screened enclosure with positive cattle in the presence of biting flies were not infected with BLV. The feeding behavior of each insect is discussed to assess their potential as vectors of BLV.