Comparison of intradermal injections of bromodeoxyuridine and tritiated thymidine in the in vivo measurement of epidermal cell turnover time in goats.

Study of keratinisation defects such as seborrhoea may require investigation of epidermal cell kinetics in clinical cases. For this to be practically useful a safe, reliable and quick method is essential. This study compared epidermal cell kinetics in the goat following the use of intradermal injection of tritiated thymidine ([3H]TdR) and bromodeoxyuridine (BURD) to label S phase nuclei. Turnover time for goat epithelial cells was not significantly different for the two agents; 26.3 +/- 6.0 days for thymidine and 21.8 +/- 5.2 days for BURD. It was concluded that intradermal injection of BURD will be a suitable technique for investigating epidermal cell kinetics in vivo and could have applications in a clinical situation because it is quicker and safer than [3H]TdR.     

Incorporation of tritiated thymidine, leucine and histidine in murine tail epidermis after skin irritation (histoautoradiography).

Thymidine incorporation into epidermal DNA as well as leucine and histidine incorporation into epidermal protein were studied by histoautoradiography in female mice tail epidermis. Prior to in vitro incubation of skin samples with labelled precursors, tail skin was irritated mechanically by rubbing with fine sand paper, chemically by repeated topical administration of n-hexadecane and by feeding an essential fatty acid deficient diet. After these skin irritations, an increased thymidine labelling index and skin thickening were found. Leucine was shown to be incorporated into protein predominantly in basal epidermal cell layers, while histidine was incorporated predominantly in the granular layer. Especially after mechanical skin irritation, this difference was obvious.     

Comparison of MTT colorimetric assay and tritiated thymidine uptake for lymphocyte proliferation assays using chicken splenocytes.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) colorimetric assay was compared with the conventional tritiated thymidine deoxyriboside (3H-TdR) incorporation for assay of lymphocyte blastogenesis using mononuclear cells isolated from the spleens of specific-pathogen-free chickens. The study was undertaken in an effort to simplify methods for assessing avian lymphocyte proliferation, specifically for evaluating response to mitogens or for indirect measurement of T-cell growth factors. The results from stimulated cells in both assay methods were significantly different from results from the control cells, and the MTT assay results regressed in a significant linear manner on counts from 3H-TdR incorporation. On this basis, the MTT assay is a valid test for evaluation of lymphocyte proliferation of chicken splenocytes.     

Efficacy of nano-hydroxyapatite prepared by an aqueous solution combustion technique in healing bone defects of goat

The present study was undertaken to evaluate porous hydroxyapatite (HAp), the powder of which was prepared by a novel aqueous solution combustion technique, as a bone substitute in healing bone defects in vivo, as assessed by radiologic and histopathologic methods, oxytetracycline labeling, and angiogenic features in Bengal goat. Bone defects were created in the diaphysis of the radius and either not filled (group I) or filled with a HAp strut (group II). The radiologic study in group II showed the presence of unabsorbed implants which acted as a scaffold for new bone growth across the defect, and the quality of healing of the bone defect was almost indistinguishable from the control group, in which the defect was more or less similar, although the newly formed bony tissue was more organized when HAp was used. Histologic methods showed complete normal ossification with development of Haversian canals and well-defined osteoblasts at the periphery in group II, whereas the control group had moderate fibro-collagenization and an adequate amount of marrow material, fat cells, and blood vessels. An oxytetracycline labeling study showed moderate activity of new bone formation with crossing-over of new bone trabeculae along with the presence of resorption cavities in group II, whereas in the control group, the process of new bone formation was active from both ends and the defect site appeared as a homogenous non-fluoroscent area. Angiograms of the animals in the control group showed uniform angiogenesis in the defect site with establishment of trans-transplant angiogenesis, whereas in group II there was complete trans-transplant shunting of blood vessel communication. Porous HAp ceramic prepared by an aqueous combustion technique promoted bone formation over the defect, confirming their biologic osteoconductive property.     

Vaccination of pigs against Aujeszky's disease by the intradermal route using live attenuated and inactivated virus vaccines.

Inactivated and live Aujeszky's disease virus vaccines were administered intradermally using a special device without a needle. The 88 pigs were vaccinated at the beginning of the fattening period, both under experimental conditions and in commercial herds. All the pigs were challenged at the end of the fattening period in isolation units. The same vaccines were also injected intramuscularly. Vaccination by the intradermal route induced good protection, similar to that conferred with live virus vaccine injected intramuscularly. The inactivated virus vaccine was not as effective when it was injected by the intradermal route. In animals vaccinated intradermally, there were no local lesions in the meat, but very small nodules were found in the dermis; these do not affect carcass quality. The effects of challenge exposure depended on the initial health of the animals, and a synthetic value (delta G) was used to interpret the data. In fattening pigs, intradermal vaccination required less animal constraint than intramuscular injection; administration could be verified by the presence of a papule at the site of inoculation, and pigs could be vaccinated while they were feeding. Injection without a needle also helps avoid bacterial contamination under farm conditions.     

Prediction of in vivo apparent total tract energy digestibility of barley in grower pigs using an in vitro digestibility technique

The DE content within cereal grains can vary 25% mainly due to changes in apparent total tract digestibility (ATTD) of energy. In vitro digestibility techniques have been developed to predict the DE value among feedstuffs. However, these techniques have not been tested properly for their suitability to predict the variation in energy digestibility and DE content within a cereal grain. The objective of the present study was to establish and evaluate an in vitro digestibility technique to predict in vivo ATTD of energy of barley in grower pigs. Barley grain samples (hulled, n = 21) with a large range in quality were collected; the ADF and CP content ranged from 4.5 to 11.4% and 10.0 to 16.4% (DM basis), respectively. The ATTD of energy was determined using barrows (n = 63, 33 +/- 2.1 kg of initial BW) in 2 periods with 6 observations per sample and ranged from 51.9 to 78.5%, with relative errors between 0.4 and 5.0%. A preliminary study, comparing a 2- and a 3-step in vitro digestibility technique using 3 barley samples, indicated that R(2) between in vivo and in vitro energy digestibility was greater using the 3- than the 2-step technique (0.92 vs. 0.76). Therefore, the 3-step in vitro digestibility technique was used solely in subsequent analyses. Briefly, ground barley was subsequently incubated with pepsin for 6 h, pancreatin for 18 h, and cellulase for 24 h. The DM and GE content of samples and residues were measured to calculate digestibility. The in vitro energy digestibility of the 21 barley samples with duplicate measurements ranged from 63.7 to 82.2%, with relative errors between 0.1 and 2.6%. In vitro energy digestibility was strongly related (y = 1.25 x - 25.22; R(2) = 0.81) to in vivo energy digestibility. Finally, a subset of 7 barley samples was analyzed in quadruplicate using the 3-step in vitro technique. The relationship between in vitro and in vivo energy digestibility was very strong (y = 1.23 x - 25.33; R(2) = 0.97) with relative errors between 0.5 and 2.7%. In vitro DE and energy digestibility were perfectly related (R(2) = 1.00). In summary, the 3-step in vitro energy digestibility technique can accurately predict the ATTD of energy in barley in grower pigs. The 3-step in vitro digestibility technique, thus, might be useful as the reference laboratory procedure to calibrate analytical equipment to rapidly predict the ATTD of energy in barley.     

Estimation of direct and social effects of feeding duration in growing pigs using records from automatic feeding stations

Automatic feeding systems in pig production allow for the recording of individual feeding behavior traits, which might be influenced by the social interactions among individuals. This study fitted mixed models to estimate the direct and social effects on visit duration at the feeder of group-housed pigs. The dataset included 74,413 records of each visit duration time (min) event at the automatic feeder from 135 pigs housed in 14 pens. The sequence of visits at the feeder was employed as a proxy for the social interaction between individuals. To estimate animal effects, the direct effect was apportioned to the animal feeding (feeding pig), and the social effect was apportioned to the animal that entered the feeder immediately after the feeding pig left the feeding station (follower). The data were divided into two subsets: “non-immediate replacement” time (NIRT, N = 6,256), where the follower pig occupied the feeder at least 600 s after the feeding pig left the feeder, and “immediate replacement” time (IRT, N= 58,255), where the elapsed time between replacements was less than or equal to 60 s. The marginal posterior distribution of the parameters was obtained by Bayesian method. Using the IRT subset, the posterior mean of the proportion of variance explained by the direct effect (Prpσ^d2) was 18% for all models. The proportion of variance explained by the follower social effect (Prpσ^f2) was 2%, and the residual variance (σ^e2) decreased, suggesting an improved model fit by including the follower effect. Fitting the models with the NIRT subset, the estimate of Prpσ^d2 was 20% but the Prpσ^f2 was almost zero and σ^e2 was identical for all models. For the IRT subset, the predicted best linear unbiased predictor (BLUP) of direct (Direct BLUP) and social (Follower BLUP) random effects on visit duration at the feeder of an animal was calculated. Feeder visit duration time was not correlated with traits, such as weight gain or average feed intake (P > 0.05), whereas for the daily feeder occupation time, the estimated correlation was positive with the Direct BLUP (r^= 0.51, P < 0.05) and negative with the Follower BLUP (r^= −0.26, P < 0.05). The results suggest that the visit duration of an animal at the single-space feeder was influenced by both direct and social effects when the replacement time between visits was less than 1 min. Finally, animals that spent a longer time per day at the feeder seemed to do so by shortening the meal length of the preceding individual at the feeder.     

Detection of rabies viral antigens in non-autolysed and autolysed tissues by using an immunoperoxidase technique

The objectives of this study were to determine the potential of an immunoperoxidase technique involving the avidin-biotin complex (ABC) stain for the diagnosis of rabies in fresh tissues and compare it with other standard methods, including the fluorescent antibody test (FAT), haematoxylin and eosin and Seller's stain, and to investigate its capacity to detect rabies antigen in autolysed tissues. Samples of non-autolysed brain from 81 domestic and wild animals suspected of having rabies were examined. Rabies antigen was detected by FAT in 41 of these samples and Negri bodies were detected in 40 (97.6 per cent) of them by the immunoperoxidase technique, in 25 by haematoxylin and eosin and in 22 by Seller's stain. The sensitivity of the immunoperoxidase technique decreased as the tissues were left to autolyse; after two days it was 91.2 per cent, after four days 70.6 per cent, and after seven days 11.8 per cent.     

Distribution of enrofloxacin and its active metabolite, using an in vivo ultrafiltration sampling technique after the injection of enrofloxacin to pigs

The objective of this study was to determine the pharmacokinetics (PK) of enrofloxacin in pigs and compare to the tissue interstitial fluid (ISF). Six healthy, young pigs were administered 7.5 mg/kg enrofloxacin subcutaneously (SC). Blood and ISF samples were collected from preplaced intravenous catheters and ultrafiltration sampling probes placed in three different tissue sites (intramuscular, subcutaneous, and intrapleural). Enrofloxacin concentrations were measured using high-pressure liquid chromatography with fluorescence detection, PK parameters were analyzed using a one-compartment model, and protein binding was determined using a microcentrifugation system. Concentrations of the active metabolite ciprofloxacin were negligible. The mean ± SD enrofloxacin plasma half-life, volume of distribution, clearance, and peak concentration were 26.6 ± 6.2 h (harmonic mean), 6.4 ± 1.2 L/kg, 0.18 ± 0.08 L/kg/h, and 1.1 ± 0.3 μg/mL, respectively. The half-life of enrofloxacin from the tissues was 23.6 h, and the maximum concentration was 1.26 μg/mL. Tissue penetration, as measured by a ratio of area-under-the-curve (AUC), was 139% (± 69%). Plasma protein binding was 31.1% and 37.13% for high and low concentrations, respectively. This study demonstrated that the concentration of biologically active enrofloxacin in tissues exceeds the concentration predicted by the unbound fraction of enrofloxacin in pig plasma. At a dose of 7.5 mg/kg SC, the high tissue concentrations and long half-life produce an AUC/MIC ratio sufficient for the pathogens that cause respiratory infections in pigs.     

The in vivo excystation of Sarcocystis gigantea and S. tenella sporocysts

In vivo studies on the excystation of Sarcocystis gigantea and S. tenella sporocysts indicated that this process was, as in vitro, a diphasic one involving both pretreatment and treatment phases. The studies also tended to support in vitro observations that the requirements for the excystation of these two species are quite different. The results suggested that for neither species was the pretreatment stimulus likely to be provided by conditions in the rumen alone. However, exposure to abomasal conditions only induced moderate levels of excystation in both when they were subsequently treated with trypsin and bile. For S. gigantea, 0.25 to 4 h abomasal exposure was most effective; for S. tenella, 24 hours. The stimuli necessary to complete the excystation process could, apparently, be provided by 1 h placement in the duodenum for S. gigantea but not for S. tenella.     

Estimation of 140S particles in foot-and-mouth disease virus (FMDV) vaccine by using the computer analyzing system

The quantity of 140S particles in inactivated foot-and-mouth disease virus (FMDV) vaccine samples produced in Foot-and-Mouth Disease Vaccine Production Center (FMD Vaccine Production Center) in Thailand was estimated by the sucrose gradient ultracentrifugation and optical density analysis by using the computer applying system. The soft ware; Chromato Data System (CDS) (Nihon Chromato Works Co., Ltd. Japan) which is prepared for the analysis of chromatography, was applied for the estimation of 140S particles in FMDV vaccine. The quantity of 140S particles in each vaccine sample measured by CDS was mostly ranged from 2-4 micrograms/ml and this quantity was consistent with the results of the other reports. This method is considered to be the available method for estimation of 140S particles in FMDV vaccine as routine assay.     

Validation of the monofrequency forced oscillation technique for pulmonary function investigation in calves: an in vitro and in vivo study.

This study was to investigate whether the monofrequency forced oscillation (MFO) technique could be accurately used for pulmonary function testing in calves. The airflow resistance measured by this technique was compared to the resistance measured by the classical reference method by an in vitro study, using an artificial lung model, and by an in vivo study, using 11 healthy calves. The effect of the reference impedance of this oscillation equipment was also investigated. The results show linear relationships (r2 > 0.924, P < 0.001) in the resistance ranges 0 to 0.7 kPa litre-1 s and 0 to 1.0 kPa litre-1 s with a reference impedance of 1.0 kPa litre-1 s and 2.0 kPa litre-1 s, respectively. The MFO resistance values lower and higher than 0.4 kPa litre-1 s are slightly overestimated and underestimated, respectively. It was shown that, if some technical requirements are met, the MFO technique is a simple, reproducible, fast and accurate method which allows measurement of airflow resistance even in field conditions.     

Assessment of pregnancy in sheep by analysis of plasma progesterone using an amplified enzyme immunoassay technique

An amplified enzyme immunoassay kit for progesterone analysis was used to diagnose pregnancy in a flock of 130 mule ewes. An accuracy of 100 per cent was obtained after the analysis of progesterone in plasma samples taken 15 to 16 days after mating. In mule ewes a plasma progesterone level greater than 5.9 nmol/litre was indicative of pregnancy. In the validation of the technique, duplicate ewe plasma samples and progesterone standards were compared with a radio immunoassay technique; the regression coefficient between the two techniques was r = 0.82.     

链烷技术估测典型草原不同放牧率绵羊食性食量方法研究

为了进一步探讨饱和链烷技术在典型草原自由放牧家畜中的应用,定量研究自由放牧绵羊食性及食量,于2004年6-9月在中国科学院内蒙古草原生态系统定位研究站放牧样地进行试验,选择体重及体况相近、健康无病的二岁羯羊(内蒙古细毛羊×蒙古羊)60只,按体重(36.9±2.6) kg聚类分组,设置6种放牧率(0,1.33,2.67,4.00,5.33和6.67羊/hm²)的轮牧实验,绵羊采食量的测定选择放牧率为1.33,4.00和6.67羊/hm²的处理,试验开始,每只羊投喂1粒QSM饱和链烷缓释胶囊。食性测定选用全部的放牧率进行,同时辅助全粪法、扣笼法一同评价。试验期内定期收集试验地牧草样品及绵羊粪便样品,使用气相色谱分析牧草和粪样中的链烷含量,应用链烷技术估测放牧绵羊的排粪量、牧草采食比例和干物质采食量。结果表明,植物链烷模式存在种间差异,运用链烷技术估测的排粪量和实际测得排粪量差异不显著(P>0.05),放牧绵羊主要采食7~9种牧草,且不同放牧季节不同放牧率绵羊采食的牧草种类和比例不同,但采食量差异不显著(P>0.05)。本研究表明,运用链烷技术结合扣笼方法可以估测天然草地自由放牧家畜牧草的采食比例、干物质采食量和排粪量。     

Estimation in vivo of the body and carcass chemical composition of growing lambs by real-time ultrasonography

The relationship between ultrasound measurements and empty body and carcass chemical composition was investigated. A 500-V real-time ultrasound with a 7.5-MHz probe combined with image analysis was used to make in vivo measurements to predict the empty body and carcass chemical composition of 31 female lambs of two genotypes, ranging in BW from 18.2 to 48.9 kg. Eleven ultrasound measurements of s.c. fat, muscle, and tissue depth were taken at four different sites (over the 13th thoracic vertebra, between the 3rd and 4th lumbar vertebrae, at the 3rd sternebra of the sternum, and over the 11th rib, 16 cm from the dorsal midline). The single best predictor of empty body fat quantity and energy value was the s.c. fat depth over the 13th thoracic vertebra (r(2) = 0.904 and 0.912; P <0.01, respectively). Body weight was used with ultrasound measurements in multiple regression equations to establish the best independent variables combination for predicting chemical composition. Results showed that BW and two of the three ultrasound measurements (s.c. fat depth over the 13th thoracic vertebra, between the 3rd and 4th lumbar vertebrae, and tissue depth over the 11th rib, 16 cm from the dorsal midline), explained 94.7 to 98.7% (P < 0.01) of the quantity of water and fat and the energy value variation in the empty body and carcass. Body weight per se was the best predictor of the quantity of protein, accounting for 97.5 and 96.8% (P < 0.01) of the variation observed in the empty body and carcass, respectively. The results of this study suggest that BW and some ultrasound measurements combined with image analysis, particularly subcutaneous fat depth over the 13th thoracic vertebra, allow accurate prediction of empty body and carcass chemical composition in lambs.     

Establishment of conditions for the detection of bovine herpesvirus-1 by polymerase chain reaction using primers in the thymidine kinase region.

Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1 (BHV-1) was developed and optimized using 22 bp sense and 20 bp antisense primers in the thymidine kinase (TK) coding region. The amplification product is 183 bp long. The PCR optimization was done using BHV-1 tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). The sensitivity of four methods of sample preparation which are standard DNA extraction, modified proteinase K (PK) digestion, GeneReleaserTM + 34 cycles or + 44 cycles, and boiling were compared with virus isolation (VI) using BHV-1TCS. The incorporation of 10% glycerol in the reaction mixture, the incubation in PK for 18 hours and predenaturation of samples and cooling in ice prior to PCR were essential for the amplification of BHV-1 DNA for samples prepared by standard DNA extraction and modified PK digestion. The preparation of samples by Gene-ReleaserTM, a proprietary nucleic acid releasing cocktail, showed 10 to 1,000-fold increase in sensitivity compared to standard DNA extraction and modified PK digestion. No amplification was observed in samples prepared by boiling. The sample preparation of BHV-1 LA strain by GeneReleaserTM showed sensitivity equivalent to virus isolation. The BHV-1 TK PCR using GeneReleaserTM has a detection limit of 1 picogram and 10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viral DNA in 1,000 infected cells in a total suspension of 10,000 cells using either ethidium bromide stained gel or Southern blot hybridization.     

Mitogen-induced transformation of sheep and goat peripheral blood lymphocytes in vitro: The effects of varying culture conditions and the choice of an optimum technique

Peripheral blood lymphocytes (PBL) prepared by centrifugation of heparinized sheep or goat jugular venous blood on Ficoll-Triosil were shown to incorporate methyl-[H³]-thymidine ([H³]-Tdr) in vitro in response to lymphocyte mitogens.Optimal conditions for transformation included the culture of 2.5 × 10⁵ viable cells per round bottomed culture well in 250μl medium RPMI-1640 supplemented with fetal calf serum (FCS) at 10% for goat or 15% for sheep lymphocytes. Optimum incorporation of [H³]-Tdr by sheep PBL was recorded after 3–5 days and was achieved in response to 100μg/ml phytohaemagglutinin (PHA), 20μl/ml pokeweed mitogen (PWM), 10μg/ml Concanavalin-A (Con-A) and 50μg/ml bacterial lipopolysaccharide (LPS). For goat PBL the optimum mitogen concentrations were 50μg/ml PHA, 20μl/ml PWM, 5μg/ml Con-A and 50μg/ml LPS. Optimum PHA concentrations were influenced by the level of FCS supplementation, higher concentrations of PHA being required for optimum response when the concentration of FCS was increased.While variability within preparations was small there was considerable variation in the magnitude of the response between preparations, which was sufficient to confound comparisons between different experiments and between animals. The variability between preparations could not be attributed to changes in sensitivity of PBL to mitogens or to the influence of erythrocyte contamination of the PBL preparations. While these results are in general agreement with previous reports of optimal conditions for the measurement of ruminant PBL to mitogens, there are some important differences which are discussed in the context of the available literature.     

Detection of Chlamydophila abortus in sheep and goat flocks in southern Italy by PCR using four different primer sets

An epidemiological survey was performed to detect the presence of Chlamydophila (C.) abortus and other members of the order Chlamydiales in ovine and caprine flocks with a history of abortion in southern Italy. Four pairs of primers were compared to evaluate their ability to detect Chlamydiales using purified DNA preparations and tissue samples from aborted foetuses with suspected chlamydial infections. As expected, amplification of DNA of the reference strain C. abortus using primer pairs U23F/23Sigr, 16SF2/23R, CTU/CTL and CpsiA/CpsiB produced fragments of about 600 bp, 585 bp, 1000 bp and 300 bp, respectively. The detection limits of the four PCR tests performed on serial DNA dilutions of the C. abortus reference strain were of 10 pg, 0.1 pg, 0.1 pg and 1 fg of DNA, respectively. The most sensitive amplification of DNA extracted from the organ tissues was obtained with primer pairs CpsiA/CpsiB, which detected Chlamydophila spp. DNA in all infected tissue samples. Only C. abortus was identified during the survey. The presence of this agent was confirmed in 3 out of 27 ovine and caprine flocks included in the survey suggesting that abortion due to C. abortus is uncommon in southern Italy.     

Detection of Chlamydophila abortus in sheep and goat flocks in southern Italy by PCR using four different primer sets

An epidemiological survey was performed to detect the presence of Chlamydophila (C.) abortus and other members of the order Chlamydiales in ovine and caprine flocks with a history of abortion in southern Italy. Four pairs of primers were compared to evaluate their ability to detect Chlamydiales using purified DNA preparations and tissue samples from aborted foetuses with suspected chlamydial infections. As expected, amplification of DNA of the reference strain C. abortus using primer pairs U23F/23Sigr, 16SF2/23R, CTU/CTL and CpsiA/CpsiB produced fragments of about 600 bp, 585 bp, 1000 bp and 300 bp, respectively. The detection limits of the four PCR tests performed on serial DNA dilutions of the C. abortus reference strain were of 10 pg, 0.1 pg, 0.1 pg and 1 fg of DNA, respectively. The most sensitive amplification of DNA extracted from the organ tissues was obtained with primer pairs CpsiA/CpsiB, which detected Chlamydophila spp. DNA in all infected tissue samples. Only C. abortus was identified during the survey. The presence of this agent was confirmed in 3 out of 27 ovine and caprine flocks included in the survey suggesting that abortion due to C. abortus is uncommon in southern Italy.