Peripartum changes in serum activities of three major alkaline phosphatase isoenzymes in Holstein dairy cows.

This study investigated changes in serum levels of hepatic, bone, and intestinal alkaline phosphatase (ALP) isoenzymes (ALP2, ALP3, and ALP5, respectively) in Holstein cows around parturition. Tartrate-resistant acid phosphatase 5b (TRAP5b) activity and calcium (Ca) concen-trations were also measured. We analyzed blood samples from 11 late-pregnant heifers (primipa-rous group) and 13 multiparous (2-4 lactations; multiparous group) cows at 3 weeks (18-24 days prepartum; -3 weeks), 2 weeks (17-11 days prepartum; -2 weeks), and 1 week (10-4 days prepar-tum; -1 weeks) before parturition; the day of calving (within 12 h post-calving; day 0); and 5 days postpartum (5 days). ALP3 activity was significantly higher in the primiparous group than in the multiparous group, whereas the activities decreased significantly in both groups after 5 days. ALP2 and ALP5 activities did not change, whereas ALP2 activity was significantly higher in the primiparous group than in the multiparous group. TRAP5b activity was significantly higher in the primiparous group than in the multiparous group and showed a transient significant increase at day 0. Ca concentration significantly decreased at day 0 in both groups; the Ca level at day 0 was significantly higher in the primiparous group than in the multiparous group. These data show that ALP3 activity in serum may indicate a change in osteoblastic bone forma-tion after calving, but further study is needed to determine the clinical application for measuring ALP isoenzymes in bovine medicine.

Agarose gel electrophoresis pattern of serum alkaline phosphatase isoenzymes in Holstein cows during lactation.

A recent study found that an agarose gel electrophoresis (AGE) method yielded two distinct major bands corresponding to the hepatic and bone ALP isoenzymes (ALP2 and ALP3, respec-tively) in bovine serum treated with protease and neuraminidase (PN-treatment), although there were concerns that the intestinal ALP isoenzyme (ALP5) often overlapped with ALP3 in human serum treated with neuraminidase. Because ALP5 was separated from ALP3 in bovine serum treated with protease alone (P-treatment), we used a modified method employing both P- and PN-treated bovine sera to measure the activities of the three ALP isoenzymes in 53 lacta-ting Holstein cows: 24 primiparous and 29 multiparous. Upon electrophoresis, 51 of 53 samples (96.2%) subjected to P-treatment yielded a distinct fraction corresponding to ALP5, as did the control serum. All PN-treated sera yielded a definite ALP2 fraction. The ALP3 fraction was calculated as the remainder after excluding ALP2 and ALP5. The activities of total ALP (t-ALP) and ALP3 in primiparous cows were higher than those in multiparous cows (p ⟨ 0.001) at early-to-peak [10-110 days in milk (DIM)] and mid (111-220 DIM) lactation. In the multi-parous cows, the ALP3 activity at late lactation (221-477 DIM) was significantly higher than that at early-to-peak lactation. Thus, the modified AGE method described here is able to discrimi-nate three fractions of ALP isoenzymes in the sera of lactating cows. The AGE pattern of circu-lating ALP isoenzymes will contribute to the understanding of the physiological bone metabolism status in lactating cows.

Determination of plasma bone-specific alkaline phosphatase isoenzyme activity in Holstein calves using a commercial agarose gel electrophoresis kit.

We measured the bone-specific alkaline phosphatase (ALP) isoenzyme activity in 67 plasma samples from 14 newborn Holstein calves using both a conventional method (featuring heat inactivation) and a commercial agarose gel electrophoresis (AGE) kit; the relevant isoenzymes were termed bone-specific ALP (BAP) and ALP isoenzyme 3 (ALP3). We explored whether the AGE kit afforded reliable data when used to analyze samples from Holstein calves. The blood was collected from the jugular vein of each calf immediately prior to the first colostrum feeding (pre-feeding), 20 and 40 h after pre-feeding, and on days 4 and 7; whereas three samples (from three calves) were not obtained. The total plasma ALP activity varied widely, exceeding the ranges of reference values. On electrophoresis, 52 of 67 plasma samples (77.6 %) clearly contained both ALP isoenzyme 2 and ALP3, as did control human serum. The total ALP activity of the 52 samples ranged from 166-1989 U/L (median: 1013 U/L), whereas the values for the other 15 samples (22.4%) exhibiting abnormal isoenzyme fractionation ranged from 1014-5118 U/L (median: 1780 U/L). In the 52 plasma samples exhibiting clearly separated isoenzymes, ALP3 and BAP activities were strongly positively correlated as revealed by Deming regression (y = 0.93x + 22.6, p⟨0.0001) and Bland-Altman analysis (ALP3/BAP activities limit of agreement: -5.1%). Thus, the AGE kit yields useful information on newborn calves, and can replace the conventional method when the total plasma ALP activity is less than approximately 1000 U/L.

Evaluation of usefulness of a commercial agarose gel electrophoresis kit (QuickGel SP) for bovine serum protein electrophoresis.

The aim of this study was to show the usefulness of a commercial agarose gel electrophoresis (AGE) kit (QuickGel SP) for separating bovine serum protein fractions in comparison with conventional cellulose acetate electrophoresis (CAE). Serum protein bands were verified using five reference reagents corresponding to albumin and α1-, ß1-, ß2-, and γ-globulins. AGE clearly revealed six separated fractions of albumin and α1-, α2-, ß1-, ß2-, and γ-globulin fractions in 100% and 77.8% in serum samples of dairy cows from the healthy (n=27) and diseased groups (n=27), respectively. The α1- and α2-globulins were not separated by CAE in 14.8% and 96.3% of the samples from the healthy and diseased groups, respectively, whereas ß2- and γ-globulin were not separated by CAE in 96.3% and 100% of the samples from the healthy and diseased groups, respectively. More than 94% of the points for the α-globulin fractions (α1- and α2-globulins), the ß-γ-globulin fractions (ß1-, ß2-, and γ-globulins), and the albumin/globulin ratio between AGE and CAE were within agreement on the Bland-Altman plots. However, the mean biases were not near zero in the albumin and ß-γ-globulin fractions. These results suggest that the high-resolution commercial AGE kit can be utilized to separate bovine serum protein fractions.

Presentation of epitope sequences from foreign viruses on the surface of apple latent spherical virus particles.

Apple latent spherical virus (ALSV) has small isometric particles that are comprised of two single-stranded RNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp20, and Vp24). We constructed ALSV vectors for presenting foreign peptides on the surface of virus particles. In these vectors, peptides can be fused to either of two C-terminal regions of Vp20 (amino acid positions between G171 and P172 or between P172 and L173) or the C-terminus (T192) of Vp24. An ALSV vector presenting the epitope sequences of the coat protein (CP) of zucchini yellow mosaic virus (ZYMV) could systemically infect host plants and was specifically recognized by antiserum against ZYMV by ELISA, immunoelectron microscopy, and immunoblotting. RT-PCR showed that the epitope sequences up to 20 amino acids were stably maintained in the chimeric ALSV for more than 10 serial passages and at least six months. Purified chimeric ALSV particles induced an immune response and the production of antibodies against ZYMV-CP in rabbits. The ALSV vector was also used for expression of an epitope from VP1 of foot-and-mouth disease virus.

Microarray-based gene expression profiling of peripheral blood mononuclear cells in dairy cows with experimental hypocalcemia and milk fever.

Although a molecular diagnostic assay using clinically accessible tissue, such as blood, would facilitate evaluation of disease conditions in humans and animals, little information exists on microarray-based gene expression profiling of circulating leukocytes from clinically hypocalcemic cows. Therefore, peripheral blood mononuclear cells from dairy cows with experimentally induced hypocalcemia or spontaneous milk fever were subjected to oligo-microarray analysis to identify specific biomarker genes. In experimental hypocalcemia induced by a 4-h infusion of 10% disodium EDTA (n=4), 32 genes were significantly up- or downregulated compared with control treatment (4-h infusion of 11% calcium EDTA; n=4). In cows with milk fever (n=8), 98 genes were expressed differentially (either up- or downregulated) compared with healthy parturient cows (n=5). From these data, the following 5 genes were selected as being strongly related to both experimental hypocalcemia and milk fever: protein kinase (cAMP-dependent, catalytic) inhibitor ß (PKIB); DNA-damage-inducible transcript 4 (DDIT4); period homolog 1 (PER1); NUAK family, SNF1-like kinase, 1 (NUAK1); and expressed sequence tag (BI537947). Another gene (neuroendocrine secretory protein 55, NESP55) was also determined to be specific for milk fever, independently of hypocalcemia. The mRNA expression of these 6 genes in milk fever cases was verified by quantitative real-time reverse-transcription PCR and was significantly different compared with their expression in healthy parturient cows. In the present study, the selected genes appeared to be candidate biomarkers of milk fever because the continuous interactions between blood cells and the entire body suggest that subtle intracellular changes occur in association with disease. However, before any genomic biomarkers are incorporated into clinical evaluation of the disease, the effect of hypocalcemia on the mRNA expression of these genes in the tissues that regulate calcium homeostasis in dairy cows should be determined.

Downregulation of serine/arginine-rich splicing factor 3 induces G1 cell cycle arrest and apoptosis in colon cancer cells.

Serine/arginine-rich splicing factor 3 (SRSF3) likely has wide-ranging roles in gene expression and facilitation of tumor cell growth. SRSF3 knockdown induced G1 arrest and apoptosis in colon cancer cells (HCT116) in association with altered expression of 833 genes. Pathway analysis revealed 'G1/S Checkpoint Regulation' as the most highly enriched category in the affected genes. SRSF3 knockdown did not induce p53 or stimulate phosphorylation of p53 or histone H2A.X in wild-type HCT116 cells. Furthermore, the knockdown induced G1 arrest in p53-null HCT116 cells, suggesting that p53-dependent DNA damage responses did not mediate the G1 arrest. Real-time reverse transcription-polymerase chain reaction and western blotting confirmed that SRSF3 knockdown reduced mRNA and protein levels of cyclins (D1, D3 and E1), E2F1 and E2F7. The decreased expression of cyclin D and E2F1 likely impaired the G1-to-S-phase progression. Consequently, retinoblastoma protein remained hypophosphorylated in SRSF3 knockdown cells. The knockdown also induced apoptosis in association with reduction of BCL2 protein levels. We also found that SRSF3 knockdown facilitated skipping of 81 5'-nucleotides (27 amino acids) from exon 8 of homeodomain-interacting protein kinase-2 (HIPK2) and produced a HIPK2 Δe8 isoform. Full-length HIPK2 (HIPK2 FL) is constantly degraded through association with an E3 ubiquitin ligase (Siah-1), whereas HIPK2 Δe8, lacking the 27 amino acids, lost Siah-1-binding ability and became resistant to proteasome digestion. Interestingly, selective knockdown of HIPK2 FL induced apoptosis in various colon cancer cells expressing wild-type or mutated p53. Thus, these findings disclose an important role of SRSF3 in the regulation of the G1-to-S-phase progression and alternative splicing of HIPK2 in tumor growth.

A single-blood-sample method using inulin for estimating feline glomerular filtration rate.

BACKGROUND: Application of a multisample method using inulin to estimate glomerular filtration rate (GFR) in cats is cumbersome. OBJECTIVES: To establish a simplified procedure to estimate GFR in cats, a single-blood-sample method using inulin was compared with a conventional 3-sample method. ANIMALS: Nine cats including 6 clinically healthy cats and 3 cats with spontaneous chronic kidney disease. METHODS: Retrospective study. Inulin was administered as an intravenous bolus at 50 mg/kg to cats, and blood was collected at 60, 90, and 120 minutes later for the 3-sample method. Serum inulin concentrations were colorimetrically determined by an autoanalyzer method. The GFR in the single-blood-sample method was calculated from the dose injected, serum concentration, sampling time, and estimated volume of distribution on the basis of the data of the 3-sample method. RESULTS: An excellent correlation was observed (r = 0.99, P = .0001) between GFR values estimated by the single-blood-sample and 3-sample methods. CONCLUSIONS AND CLINICAL IMPORTANCE: The single-blood-sample method using inulin provides a practicable and ethical alternative for estimating glomerular filtration rate in cats.

Estimation of glomerular filtration rate in rabbits by a single-sample method using iodixanol.

To estimate the glomerular filtration rate (GFR) in conscious rabbits, a single-sample method using the non-ionic contrast medium iodixanol was compared with a three-sample method using the standard agent inulin. Iodixanol and inulin were co-administered intravenously to male New Zealand White rabbits at 60 mg I/kg and 40 mg/kg, respectively, and blood was collected 30, 60, 90 and 120 min later. Serum iodixanol and inulin concentrations were separately determined by high performance liquid chromatography and colorimetry, respectively. Serum urea nitrogen (UN) and creatinine concentrations were also determined. Based on the data from healthy and cisplatin-treated rabbits, the GFR estimated by iodixanol was well consistent with that by inulin. Further, when the GFR decreased to more than 60% of the reference value, serum creatinine concentrations became elevated. However, serum UN concentrations exhibited wide fluctuations, presumably due to a difference in renal handlings. The single-sample method using iodixanol was considered to be an expedient tool in both clinical and research settings, because the stress due to a multi-sample method was reduced.

Effects of cortisol secreted via a 12-h infusion of adrenocorticotropic hormone on mineral homeostasis and bone metabolism in ovariectomized cows.

To evaluate the effects of endogenously secreted cortisol on mineral homeostasis and bone metabolism in cows, 4 ovariectomized Holstein cows were infused for 12 h with either an adrenocorticotropic hormone (ACTH) solution (0.5 mg/2 L isotonic NaCl solution per cow) or isotonic NaCl solution in a 2×2 crossover design. ACTH infusion stimulated cortisol secretion and increased plasma cortisol concentrations for 18 h (P<0.001), leading to an elevated plasma glucose concentration until 36 h (P<0.001). Plasma calcium and magnesium concentrations in ACTH-infused cows fluctuated within normal ranges, whereas hypophosphatemia was observed unequivocally. The biochemical bone resorption markers tartrate-resistant acid phosphatase 5b and hydroxyproline decreased following ACTH infusion (P<0.001 and P=0.003, respectively). Similarly, the bone formation marker, bone-specific alkaline phosphatase, decreased continuously until 72 h after the ACTH infusion (P<0.001). These results demonstrate that increased secretion of cortisol via a 12-h ACTH infusion disrupted homeostasis of inorganic phosphate and suppressed bone metabolism in ovariectomized cows without involving gonadal steroid hormones.

Sensitive test for screening for Staphylococcus aureus in bovine mastitis by broth cultivation and PCR.

An assay was developed and evaluated for screening for Staphylococcus aureus in milk samples from cases of bovine mastitis by overnight cultivation in a broth containing 7.5 per cent sodium chloride, followed by pcr to amplify the nuc gene. The assay could detect concentrations of S aureus as low as 1 colony-forming unit/ml milk. Among 106 milk samples collected from individual quarters of lactating cows in one dairy herd and from a bulk tank, S aureus was detected in nine samples by the pcr assay but in only three samples by conventional microbiological culture.

Dose response to vaginal administration of 1,25-dihydroxyvitamin D3 to cows.

It was previously reported that intravaginal (IVAG) administration of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) might be protective against bovine hypocalcaemia. In the present study, various doses of exogenous 1,25(OH)(2)D(3) were administered IVAG to ovariectomised cows, and the subsequent changes in the biochemical parameters of the blood were measured to assess the characteristics of vaginal absorption. Five cows received 1,25(OH)(2)D(3) IVAG at a dose of 0.125, 0.25, 0.5, or 1.0microg/kg of body weight (BW) or intravenously at a dose of 1.0microg/kg BW. Dosing was at intervals of at least two weeks in a 5x5 Latin square design. Vaginally administered 1,25(OH)(2)D(3) was absorbed in a dose-dependent manner. There was no correlation between the IVAG dose of 1,25(OH)(2)D(3) and subsequent changes in plasma calcium concentrations. The bioavailability of 1,25(OH)(2)D(3) administered IVAG at 1.0microg/kg BW was approximately 93%.

The expression of genes for transepithelial calcium-transporting proteins in the bovine duodenum.

Intestinal epithelial cells contain calcium-binding proteins and Ca2+-transporting adenosine triphosphatase (Ca2+-ATPase), which play important roles in intestinal Ca transport. However, the factors that affect the expression of these transepithelial Ca-transporting proteins in dairy cattle are unknown. In this study, a semi-quantitative reverse transcription polymerase chain reaction was used to determine the expression of the mRNAs for intestinal Ca-binding protein calbindin-D9k (CaBP9k), two isoforms of plasma membrane Ca2+-ATPase (PMCA1 and PMCA4), and vitamin D receptor (VDR) in duodenal tissue samples from 20 non-lactating, non-pregnant Holstein dairy cattle (0.4-135.9 months old). The correlations between the expressions of transepithelial Ca-transporting proteins, the ages of the cattle, and the presence of several plasma components were evaluated. The duodenal CaBP9k mRNA content had a significant negative correlation with age and positive correlations with plasma inorganic phosphorus (iP) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) concentrations. The PMCA1 mRNA content was negatively correlated with the plasma Ca concentration. The duodenal PMCA4 mRNA content was correlated negatively with the plasma iP. The VDR mRNA content had a positive correlation with the plasma magnesium concentration.

Distribution of new methylene blue injected into the caudal epidural space in cattle.

To investigate the distribution of solutions injected into the first intercoccygeal epidural space, 24 adult, standing cattle were randomly assigned to 5-, 10- and 20-mL groups and injected with 0.12% new methylene blue (NMB) in 0.9% saline. Ten heifers received 1 mL NMB solution/100 kg of body weight. There was a significant correlation between the injected volume and the number of cranially stained spinal segments in three adult cattle groups (correlation coefficient R2=0.46; P<0.0001). In three cattle, NMB solution did not distribute more than one spinal segment cranially from the injection site due either to fibrosis of the epidural tissue or to inadvertent intravenous administration into the epidural vein. The study showed that the larger the volume of solution injected, the greater the spread with increased individual variation. The results could form the basis for determining the volume of injection required and for evaluating the pharmacokinetics of anaesthetics used in caudal epidural anaesthesia.

Technical note: Vaginal absorption of 1,25(OH)2D3 in cattle.

Although exogenous 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] administered via intravenous, intramuscular, and oral routes has been tested for efficacy in preventing parturient hypocalcemia in dairy cows, there are few reports concerning intravaginal administration. For this study, 1,25(OH)2D3 was administered via the bovine vaginal lumen, and subsequent changes in blood chemistry, including levels of 1,25(OH)2D3 and several minerals, were measured to confirm vaginal absorption. Each of 5 heifers received a single intravaginal dose of 1 microg of 1,25(OH)2D3/kg body weight; a single control heifer received the ethanol carrier alone. In heifers receiving 1,25(OH)2D3, the plasma 1,25(OH)2D3 levels increased markedly from baseline (88.3 +/- 20.3 pg/mL) within 2 h and reached a peak at 6 h after treatment (1967.4 +/- 1139.6 pg/mL). Plasma Ca levels increased from baseline (10.4 +/- 0.4 mg/dL) to a peak of 11.96 +/- 0.7 mg/dL at 24 h. The levels of inorganic phosphate in plasma increased over time from 7.3 +/- 0.5 to 8.1 +/- 0.8 mg/dL by 6 h and were maintained at a plateau level (9.1 +/- 0.7 to 8.6 +/- 0.6 mg/dL) from 24 to 96 h after treatment. Plasma magnesium decreased from a baseline level of 2.1 +/- 0.1 mg/dL to a plateau level of 1.8 +/- 0.1 mg/dL, which was sustained from 24 to 48 h after treatment. The present study provides evidence of the absorption of exogenous 1,25(OH)2D3 from the bovine vaginal wall, as shown by the marked elevation of plasma 1,25(OH)2D3 levels by 2 h after administration, and indicates the possible utility of intravaginal administration of 1,25(OH)2D3 for prophylaxis of hypocalcemia.

Comparison of xylazine, lidocaine and the two drugs combined for modified dorsolumbar epidural anaesthesia in cattle.

Twenty-four Holstein cattle scheduled for flank surgery in a standing position were randomly assigned to four groups of six. A 16 G, 120 mm Tuohy needle was inserted into the first interlumbar epidural space and its position was confirmed by the hanging drop technique. After air had been allowed to enter freely for approximately one minute, the epidural needle was slowly inserted 7 to 10 mm deeper to penetrate the epidural fat, and anaesthetic solution containing either 0.05 mg/kg bodyweight xylazine hydrochloride (xylazine), 0.025 mg/kg xylazine, 0.025 mg/kg xylazine and 0.1 mg/kg lidocaine hydrochloride (lidocaine), or 0.2 mg/kg lidocaine alone was administered. Signs of sedation were observed in the three groups treated with xylazine and the number of spinal segments involved in the area of analgesia when the anaesthetic contained xylazine was significantly greater than with 0.2 mg/kg lidocaine alone ( < 0.01). After the treatment with 0-025 mg/kg xylazine and 0.1 mg/kg lidocaine, flank surgery was performed successfully without additional line block or side effects.

Comparative histology of the laminar bone between young calves and foals.

Laminar bone or primary plexiform tissue, not Haversian bone, shows an alternative concentric pattern of laminar-bone units or plates around the bone marrow periphery of long bones, although the laminar bone is gradually replaced by osteons during the growth period. One laminar-bone unit is constructed with a hypercalcified line in the center, woven bone on both sides of the line, and lamellar bone with laminated appositional lines. Such a laminar bone showing a homogeneous calcification has been reported in young calves and some young large animals, but it has not been reported in foals although a previous report proposed that the bone structure was distinguishable from plexiform tissue. In this study, we compared young calves with foals by backscattered electron imaging mainly of transverse ground sections of mid-diaphysis. Foals had many hypercalcified lines arranged concentrically around the bone marrow periphery, which were similar to those of young calves. However, rows of cylindrical osteon-like structures with Haversian canal-like canals running along the long-bone axis were arranged between the concentric hypercalcified lines. Each Haversian canal-like structure was enclosed with laminated appositional rings of lamellar bone deposited on the woven bone. In the developing period, the bone units containing the concentric hypercalcified lines were basically equal to the laminar-bone units. The osteon-like structures or 'pseudo-osteons' were gradually replaced by 'true osteons' during the growth period. The blood vessels in the Haversian canal-like canals of foals ran along the long-bone axis, whereas the blood vessels in the concentrically prolonged bone cavities of young calves ran transversely to obliquely against the long-bone axis. Thus, the long-bone cortex of foals showing an alternative concentric pattern of a row of the osteon-like structures arranged between the hypercalcified lines will be histologically classified into a variety of laminar bone caused by the different arrangement of blood vessels. Such a laminar bone may have a biomechanical structure against physical stress, especially the modified laminar bone of foals with osteon-like structures, when compared with the typical concentric laminar bone of young calves and also Haversian bone possessing variously calcified numerous osteons caused by bone remodeling.

Characterization of the small subgenomic RNA of Soybean dwarf virus.

We have characterized a small subgenomic RNA of Japanese strains of Soybean dwarf virus (SbDV). Northern blot analyses of SbDV-infected plants showed that the small RNA contained the 3' terminal sequence of the genome and was detected in four typical Japanese SbDV strains, YS, YP, DS and DP. In the case of SbDV-DS, the RNA was 220 nucleotides in length and was transcribed from the 3' terminal region of the genome. This RNA appeared at a similar time to genomic RNA and a large sgRNA, and thereafter persisted in the infected plant. Since no conserved open reading frame (ORF) among the strains was postulated in the 3' terminal region, the small subgenomic RNA may have some regulatory roles in SbDV infections.

Antagonistic effects of intravenous or epidural atipamezole on xylazine-induced dorsolumbar epidural analgesia in cattle.

This study was performed to clarify the antagonistic actions of intravenous or epidural atipamezole on the sedative and analgesic effects of xylazine administered between the epidural fat and dura mater through the first interlumbar space in cattle. Cattle received 5 mL of a solution containing 0.05 mg x kg(-1) xylazine in 0.9% saline. Thirty minutes later, 5 mL of 0.9% saline was administered through the same needle (treatment 1) (XSE). In treatments 2 (XAE) and 3 (XAV), 5 mL of a solution containing 0.025 mg x kg(-1) atipamezole in 0.9% saline was administered epidurally or intravenously, respectively. Sedation and analgesia were similar in all three treatment groups and could be reversed by atipamezole given by either route. In the XAV treatment, the flank area relapsed into analgesia 25+/-5.8 min following reversal of the analgesic effect, and was maintained for 112.5+/-63.8 min. The present study confirmed that the sedative and analgesic effects of xylazine are completely reversed by atipamezole and can be influenced by the epidural fat in cattle. Furthermore, it seems probable that analgesia following epidural administration of xylazine is mediated by alpha(2)-adrenergic receptors, not by a local anaesthetic effect.