To test the effect of mammary blood flow on net uptakes of milk precursors by the mammary glands, inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX) were infused into the mammary circulation of 4 lactating cows. Inhibitors were infused in a 4×4 Latin square design, where treatments were infusion for 1 h of saline, NOS inhibitor (Nω-nitro-l-arginine methyl ester hydrochloride), COX inhibitor (indomethacin), or both NOS + COX inhibitors into one external iliac artery. Para-aminohippuric acid was also infused to allow for estimation of iliac plasma flow (IPF), of which approximately 80% flows to the mammary glands. Blood samples were collected before, during, and after inhibitor infusion from the contralateral external iliac artery and ipsilateral mammary vein. Inhibition of COX and NOS each produced a decrease in IPF, although the NOS effect was smaller and IPF continued to be depressed throughout the recovery period. The combination of COX and NOS inhibition produced a 50% depression in IPF and there was no carryover into the recovery period. Treatments that depressed IPF also increased arterial concentrations of acetate, β-hydroxybutyrate (BHBA), and glucose. Similarly, arteriovenous differences of acetate, BHBA, and glucose were all increased during IPF depression. To correct for a potential effect of arterial concentration, arteriovenous differences were normalized to arterial concentration, producing an extraction percentage. Inhibition of COX increased glucose extraction and tended to increase acetate and BHBA extraction. Dual inhibition only increased BHBA extraction and had no effect on mammary extraction of other metabolites. These extractions did not increase because clearances of glucose and TAG decreased as IPF decreased, and clearances of acetate and BHBA tended to decrease. Net uptake of TAG was depressed by dual NOS/COX inhibition, whereas uptakes of acetate, BHBA, and glucose were not affected by any of the treatments. To separate effects of flow from effects of arterial concentration, uptakes were regressed against IPF and arterial concentration simultaneously. According to the slopes of the regressions, a 10% decrease in IPF from the mean observed during saline infusion resulted in 3.8, 7.3, and 10.4% decreases in uptakes of acetate, glucose, and triacylglycerol, respectively. These findings indicate that mammary blood flow affects milk precursor uptake, and that clearance should not be assumed constant to predict mammary uptakes of milk precursors in situations where blood flow is changing.

Purpose: Electroarthrography (EAG) is a new technology that non-invasively measures cartilage streaming potentials through electrodes contacting the skin surrounding an articular joint. Streaming potentials are produced during cartilage compression and directly reflect cartilage composition, structure and load bearing properties. Study objectives were to develop an approach for measuring EAG in the fetlock joints of live horses, as well as to compare externally measured EAG to direct measurements of cartilage quality. Methods: EAG was performed on both forelimb fetlock (metacarpophalangeal) joints of three horses, aged 7, 9 and 16 years, following completion of a comprehensive lameness examination and radiographic assessment. For EAG, gold-plated disk electrodes were attached to skin at four sites around the fetlock, the medial (EAG1) and lateral (EAG2) anterior (dorsal) phalanx/cannon interface, as well as the medial (EAG3) and lateral (EAG4) phalanx/cannon interface (Fig. 3). Horses were positioned with the instrumented forelimb on a force plate (Kistler 9281B) and EAG signals collected wirelessly during loading. Loading was achieved by manually lifting the contralateral forelimb, which caused a shift in body weight to the instrumented forelimb, holding it aloft for 5 seconds, and releasing. EAG coefficients (μV/kg) were calculated for each electrode by fitting EAG signals to axial loads. One horse (16 year old) was euthanized for unrelated reasons and the distal forelimbs, comprising the third metacarpus (cannon bone) to the hoof, collected for in vitro tests. Each forelimb was mounted in a servohydraulic mechanical tester (Instron 8800) and EAG measured during simulated loading. Joints were then disarticulated and direct measurements of cartilage streaming potentials made at 250 sites per joint with the Arthro-BST, an arthroscopic device that non-destructively measures streaming potentials by compressing cartilage with a hemispherical indenter containing an array of 37 microelectrodes. The Arthro-BST calculates a quantitative parameter (QP) corresponding to the number of microelectrodes in contact with cartilage when the sum of streaming potentials reaches 100 mV. Results: EAG coefficients for anterior electrodes ranged from 1.51 to 2.30 μV/kg and 0.74 to 2.50 μV/kg for EAG1 and EAG2, respectively, and -2.14 to -0.42 μV/kg and -1.67 to 0.04 μV/kg for medio-lateral electrodes EAG3 and EAG4, respectively. All three horses displayed mild to moderate lameness, with the 16 year old former racehorse exhibiting significant swelling of the left fetlock and a bowed tendon at the third metacarpus. No radiographic evidence of joint disease was detected in these horses. Similar loading profiles were observed during in vivo and in vitro EAG tests and streaming potentials at anterior electrodes (EAG 1, EAG 2) closely followed axial loads (Fig. 1). A strong and significant correlation (r = 0.804, p = 0.16, n = 8) was detected between in vivo and in vitro EAG coefficients when calculated with respect to a reference electrode placed on bone beneath the articulation (Fig. 2). Direct measurements of cartilage streaming potentials revealed elevated QP, indicating lower cartilage stiffness (Fig. 3), compared to normal cartilage measured previously, which had average QP of 6.8 ± 1.9 (n = 418) and 5.6 ± 1.9 (n = 561) in the phalanx and cannon, respectively. Conclusions: EAG was measured successfully in live horses during loading with the instrumented forelimb positioned on a floor-mounted force plate. EAG coefficients acquired in vivo from a 16 year old horse correlated strongly with those obtained during analogous in vitro EAG tests on the same fetlocks (Fig. 2). Direct cartilage assessment using the Arthro-BST device revealed mild to moderate cartilage degeneration although this horse was radiographically normal. This study demonstrated the application of EAG, a novel, non-invasive cartilage assessment method, in a clinical setting. EAG may be more sensitive to early cartilage degeneration than radiography and may contribute an objective measure of cartilage quality that could enhance a typical lameness examination.Figure 2Pearson's correlation (r = 0.804, p = 0.16) between EAG coefficients at four electrodes per fetlock obtained during in vivo and in vitro assessments in a 16 years old horse. For this analysis, EAG coefficients were referenced to an electrode placed on bone beneath the articulation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3QP maps illustrating direct measurements of cartilage streaming potentials on the articular surfaces of the fetlock joints of the 16 year old horse that underwent both in vivo and in vitro EAG testing. Approximate locations of EAG electrodes placed on skin external to the articulation are identified as medial anterior phalanx/canon interface (EAG1), lateral anterior phalanx/canon interface (EAG2), medial medio-lateral phalanx/canon interface (EAG3) and lateral medio-lateral phalanx/canon interface (EAG4). QP is inversely proportional to cartilage stiffness so that higher QP represents softer or degraded cartilage. QP of normal cartilage obtained in earlier experiments (data not shown) were 6.8 ± 1.9 (n = 418) for the phalanx and 5.6 ± 1.9 (n = 561) for the cannon. Images of right joint surfaces are inverted to facilitate left/right comparisons.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

To test which, if any, of the major milk precursors can elicit a rapid change in the rate of mammary blood flow (MBF) and to define the time course and magnitude of such changes, 4 lactating cows were infused with glucose, amino acids, or triacylglycerol into the external iliac artery feeding one udder half while iliac plasma flow (IPF) was monitored continuously by dye dilution. Adenosine and saline were infused as positive and negative controls, respectively, and insulin was infused to characterize the response to a centrally produced anabolic hormone. To test the roles of cyclooxygenase, NO synthase and ATP-sensitive K (KATP) channels in nutrient-mediated changes in blood flow, their respective inhibitors-indomethacin, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), and glibenclamide-were infused simultaneously with glucose. Each day, 1 infusate was given twice to each cow, over a 20-min period each time, separated by a 20-min washout period. In addition, each treatment protocol was administered on 2 separate days. A 73% increase in IPF during adenosine infusion showed that the mammary vasodilatory response was quadratic in time, with most changes occurring in the first 5min. Glucose infusion decreased IPF by 9% in a quadratic manner, most rapidly in the first 5min, indicating that a feedback mechanism of local blood flow control, likely through adenosine release, was operative in the mammary vasculature. Amino acid infusion increased IPF 9% in a linear manner, suggesting that mammary ATP utilization was stimulated more than ATP production. This could reflect a stimulation of protein synthesis. Triacylglycerol only tended to decrease IPF and insulin did not affect IPF. A lack of IPF response to glibenclamide indicates that KATP channels are not involved in MBF regulation. Indomethacin and L-NAME both depressed IPF. In the presence of indomethacin, glucose infusion caused a quadratic 9% increase in IPF. Indomethacin is an inhibitor of mitochondrial function, so the glucose-induced increase in IPF was interpreted as feedback on mammary adenosine release from an anabolic response to glucose. Because NO synthase was not inhibited during indomethacin infusion, the feedback system is postulated to act through endothelial NO synthase. In the presence of L-NAME, glucose infusion had no effect on IPF, indicating that endothelial cyclooxygenase is not involved in glucose-induced changes in MBF.

REASONS FOR PERFORMING STUDY Artefacts caused by regional anaesthesia can influence image interpretation of ultrasonography and nuclear scintigraphy. Perineural and intrasynovial anaesthesia are commonly performed prior to magnetic resonance imaging (MRI); and the effects on MR images, if any, are unknown. OBJECTIVES To determine if perineural and intrasynovial anaesthesia of structures in the equine foot cause iatrogenic changes detectable with MRI. METHODS A baseline MRI examination of both front feet was performed on 15 horses, 2-6 days prior to mepivacaine injection adjacent to the lateral and medial palmar digital nerves, and into the podotrochlear bursa, digital flexor tendon sheath and distal interphalangeal joint of one randomly assigned forelimb. Magnetic resonance imaging was repeated at 24 and 72 h post injection; then qualitative and quantitative assessments of MRI findings were performed. RESULTS Magnetic resonance imaging findings associated with the palmar digital nerves, podotrochlear bursa and distal interphalangeal joint at 24 and 72 h after mepivacaine injection did not alter significantly from those at baseline. Compared with baseline, a significant increase in synovial fluid volume of the digital flexor tendon sheath was detected with MRI at 24 and 72 h post injection. CONCLUSIONS Perineural anaesthesia of the palmar digital nerves and intrasynovial anaesthesia of the podotrochlear or distal interphalangeal joint did not interfere with the interpretation of MR images acquired at 24 or 72 h after injection. However, intrasynovial anaesthesia of the digital flexor tendon sheath caused an iatrogenic increase in synovial fluid, detectable on MR images for at least 72 h. POTENTIAL RELEVANCE Although a definite time frame for resolution of digital flexor tendon sheath distension was not determined, we recommend waiting more than 3 days between intrasynovial anaesthesia of the digital flexor tendon sheath and evaluation with MRI.

Low concentrations of the essential amino acid histidine in circulation have been shown to increase mammary blood flow and it has been suggested that this effect is mediated by histamine. The hypotheses tested in this experiment were that interstitial histamine concentrations in the mammary gland are related to arterial His concentrations and that mammary blood flow is reduced by extracellular histamine via H(1) receptors. The hypotheses were tested by infusing saline or chlorpheniramine, a blocker of the H(1) histamine receptor, into the arterial supply of the mammary glands of lactating cows infused with 44 g/h of amino acid mixtures with or without His for 10 h. Infusates were administered in a 2 x 2 factorial arrangement within a 4 x 4 Latin square to 4 multiparous Holstein cows in mid lactation. Exclusion of His from the infusate decreased protein content in milk from the infused udder half from 3.98 to 3.77%, and increased arterial alpha-aminonitrogen concentration from 3.2 to 3.4 mM. Neither the decreased arterial His concentration nor the H(1) blocker affected plasma flow to the infused udder half. We conclude that histamine is not involved in the regulation of mammary blood flow. The H(1) blocker decreased milk production in the infused udder half from 4.6 to 3.5 kg without affecting protein, fat, and lactose percentages, suggesting an inhibition of milk ejection. Cows on chlorpheniramine ate less feed during the infusion than saline-infused cows, which resulted in lower arterial concentrations and mammary uptakes of acetate. The efficiency of plasma triacylglycerol uptake across the mammary glands was decreased by chlorpheniramine but net uptake of long-chain fatty acids was not affected. The mechanism by which an amino acid deficiency influences mammary blood flow does not involve histamine signaling through the H(1) receptor and remains unidentified.

Rates of secretion of components into milk are a function of precursor concentrations and parameters that describe expression of the milk synthetic enzymes and their sensitivity to precursor concentrations. To establish the enzymatic sensitivities of milk fat yield and mammary acetate utilization to circulating acetate concentration, lactating cows were infused for 10 h with 0 or 40 g of acetate/h in an external iliac artery supplying one udder half. In addition, to investigate the possibility that energy supply influences the milk protein response to an elevated amino acid (AA) concentration, 2 different AA profiles were infused with and without acetate. Six cows, fed a total mixed ration of 21% crude protein ad libitum, were infused with AA at 0 g/h, 30 g/h in the profile of rumen microbes, or 30 g/h in the profile of milk proteins, in a 3 x 2 factorial arrangement with the 2 acetate treatments of 0 and 40 g/h, all in a 6 x 6 Latin square. Amino acid infusion caused a 60% increase, on average, in plasma concentration of AA entering the infused udder half. From the microbial AA profile, 49% of infused AA were taken up by the udder half, 42% of which occurred during the first pass. From the milk AA profile, 44% of infused AA were taken up by the udder half, 50% of which occurred during the first pass. There was an 8% increase in yield of milk protein with AA infusion, representing 7% capture, but no effect of the infused profile. Acetate infusion caused a decrease in the yields of milk protein and lactose when AA were infused, but not when AA were absent. Milk fat yields were not affected, although acetate concentrations in plasma entering the infused udder half increased by 123% and mammary uptakes increased by 128%. Mammary uptakes of long-chain fatty acids and beta-hydroxybutyrate were not affected by acetate infusion, whereas glucose uptakes tended to increase. It was suggested that excess acetate may have been sequestered in adipose tissue in the udder. Yields of both protein and fat in milk showed a low sensitivity to the concentration of their precursors in circulation. It was concluded that the Km in Michaelis-Menten-type equations describing milk synthesis should be assigned a low value, and that the Vmax is regulated to bring about changes in milk yield and composition.

To quantify kinetics of mammary glucose utilization in vivo, 24 paired glucose and extracellular indicator (p-aminohippuric acid) dilution curves across intact bovine mammary glands were obtained after bolus injections into the external iliac artery. Dilution curves were analyzed using a compartmental capillary, convolution integration model. Four candidate submodels of glucose transport and metabolism in capillary supply zones were fit to the glucose dilution curves and evaluated. Model I, with one extracellular compartment for glucose and first-order unidirectional uptake, failed, indicating that efflux of glucose from the intracellular space could not be ignored. Model II, with first-order exchanges between extracellular and intracellular compartments and sequestration from the latter, was overdefined because unidirectional clearance of glucose was at least five times the blood flow rate and 20 times the net clearance rate. Model III, combining extracellular and intracellular space into one compartment, was superior in its goodness-of-fit to curves and identifiability of parameters. Michaelis-Menten parameters of sequestration were not identifiable. Parameters of the optimal compartmental capillary, convolution integration model were applicable to both the dynamics of injected glucose dilution and the steady-state background arteriovenous difference of glucose. Glucose sequestration followed first-order kinetics between 0 and 7 mM extracellular glucose with an average rate constant of 0.006 s(-1) or a clearance of 44 ml/s. The ratio of intracellular to extracellular glucose distribution space was 0.34, which is considerably lower than the expected intracellular volume and suggests an intracellular occlusion compartment with which extracellular glucose rapidly exchanges.

Thirty-three paired indicator/nutrient dilution curves across the mammary glands of four cows were obtained after rapid injection of para-aminohippuric acid (PAH) plus glucose into the external iliac artery. For the measurement of extracellular volume and kinetics of nutrient uptake from indicator dilution curves, several models of solute dispersion and disappearance have been proposed. The Crone-Renkin models of exchange in a single capillary assume negligible washout of solutes from the extracellular space and do not describe entire dilution curves. The Goresky models include a distribution of capillary transit times to generate whole system outflow profiles but require two indicators to parametize extracellular behavior. A compartmental capillary, convolution integration model is proposed that uses one indicator to account for the extracellular behavior of the nutrient after a paired indicator/nutrient injection. With the use of an iterative approach to least squares, unique solutions for nonexchanging vessel transit time t(mu) and its variance sigma were obtained from all 33 PAH curves. The average of heterogeneous vascular transit times was approximated as 2sigma = 8.5 s. The remainder of indicator dispersion was considered to be due to washout from a well-mixed compartment representing extracellular space that had an estimated volume of 5.5 liters or 24% of mammary gland weight. More than 99% of the variation in the time course of venous PAH concentration after rapid injection into the arterial supply of the mammary glands was explained in an unbiased manner by partitioning the organ into a heterogeneous nonexchanging vessel subsystem and a well-mixed compartmental capillary subsystem.

The effect of jugular infusions of glucose and amino acids on activation of p70S6K was studied in the bovine mammary gland.Cows were starved for 22 h to quench milk protein synthesis before resupplying EEA+Glc, Glc, Met+Lys, His, Leu or saline.Results implicate glucose as a regulator of global, rather than ribosomal, protein synthesis.EAA appear to play a far greater role in p70S6K regulation than glucose.His and Leu appear to act as metabolic agents in the mammary epithelial cell; Leu through elevating RNA levels thereby contributing to the long-term protein synthesis capacity, and His through stimulating protein translation.

Concentrations of glucose in the external iliac artery feeding one udder half of 14 midlactation Holstein cows were increased by infusion to test the following three hypotheses of mammary function: 1) that mammary glands control their blood supply to maintain intracellular energy balance, 2) that milk precursors are taken out of capillary blood according to mass action kinetics, and 3) that the rate of milk component synthesis is dependent on its precursor's uptake from blood.The first seven cows received 20 g/h glucose during 10 h of infusion.Arterial concentrations of glucose were locally increased by only 10%, and the iliac plasma flow was not affected by glucose infusion, so the next seven cows were given 90 g/h glucose.Quantitative predictions resulting from the hypotheses were that arterial plasma flow would decrease by 32% with 90 g/h glucose infusion, glucose uptakes would increase and acetate, fatty acid, and amino acid uptakes decrease, and milk protein and fat yields and percentages would decrease.Iliac plasma flow decreased 16%, half of what was predicted, which suggests that other regulatory processes besides blood flow control took part in the response.Acetate and fatty acid uptakes by the mammary glands were reduced as predicted because of the lower blood flow, but an unexpected depression in extraction of plasma triacylglycerol also contributed to the reduced fatty acid uptake.Milk fat and protein yields were not affected by the exogenous glucose, falsifying the third hypothesis that milk component secretion is a function of uptake of its precursor.Milk fat and protein percentages declined with glucose infusion because of increased lactose synthesis and secretion of water into milk.

To evaluate a close mammary infusion technique for the study of milk protein responses to blood amino acid profile, five early-lactation, multiparous Holstein cows were surgically fitted with catheters in both external iliac arteries. Animals were infused into one arterial catheter with five different solutions on 5 consecutive days in a Latin square design. Infusions began at 0800 h and continued until 1800 h. The five infusates were a 3% saline control, 15 g/h of complete amino acid mix, 15 g/h of imbalanced amino acid mix (minus His), 30 g/h of complete amino acid mix, and 30 g/h of imbalanced amino acid mix (minus His). Cows were fed a total mixed ration twice daily containing 16% crude protein and 1.7 Mcal/kg of net energy for lactation. Infusion of the complete amino acid mix elevated amino acid concentrations in arterial plasma two- to threefold but caused only a small dose-dependent increase in milk protein content and yield. Fat percentage in milk was decreased from 4.08 to 3.35% by the complete amino acid infusions so that the protein:fat ratio climbed from 0.76 on the control to 0.99 with 30 g/h of amino acid. Removal of His from the infusate caused plasma His concentrations to drop but had no effect on any other circulating amino acids. Milk composition was restored to control levels by removal of the single amino acid. A short-term circulating amino acid imbalance depresses milk protein percentage and increases milk fat content in dairy cows.

SummaryAs orthopaedic investigations have become more intricate, bone specimens have sometimes undergone multiple freeze-thaw cycles prior to biomechanical testing. The purpose of this study was to determine if repeated freezing and thawing affected the mechanical properties of canine cortical bone. Six pairs of third-metacarpal bones were tested in three-point bending and six pairs of femurs were tested in torsion. At the time of collection, one member of each pair was tested destructively. The other member was tested nondestructively at the time of collection and after each of five freeze-thaw cycles, followed by destructive testing after the fifth cycle. For destructive tests, the material properties (modulus, maximum stress, maximum strain and absorbed energy) of a specimen at the time of collection were compared to those of the corresponding contralateral specimen that had undergone five freeze-thaw cycles. For repeated nondestructive tests, the modulus of a specimen at the time of collection was compared to modulus of the same specimen at each of the five thaw intervals. During destructive testing, there was a significant (p = 0.02) decrease (20%) in maximum torsional strain. Other changes in bending and torsional destructive properties were not statistically significant. During repeated nondestructive testing, there were solitary significant (p < 0.05) increases (8% and 9%, respectively) in both bending and torsional modulus. However, these isolated changes were not correlated to the number of freeze-thaw cycles. The pattern of alterations in destructive and non-destructive biomechanical properties was most consistent with varying specimen dehydration at each thaw interval. Despite using accepted methods to maintain specimen hydration, repeated freezing, thawing, handling and testing of cortical bone increased the risk of moisture loss. Unless stringent efforts are made to ensure proper hydration, the mechanical properties of canine cortical bone will be altered by repeated freezing and thawing, affecting the results of studies utilizing this technique.The effect of five freeze-thaw cycles on paired canine cortical bone specimens was evaluated using destructive and repeated non-destructive three-point bending and torsion tests. A significant decrease in destructive torsional strain and isolated significant increases in nondestructive bending and torsional modulus were most consistent with varying specimen dehydration at each thaw interval.

Binding between equine plasma alpha-2-macroglobulin (alpha(2)M) and several cytokines known to participate in inflammatory reactions in other species was initially examined. Plasma was obtained from 5 horses with various abnormalities. Samples, both untreated and after reaction with methylamine, were incubated with exogenous, radiolabeled, porcine-derived transforming growth factor-beta-1 (I-125-TGF-beta 1), recombinant human interleukin-1-beta (I-125-IL-1 beta), and recombinant human tumor necrosis factor-alpha (I-125-rhTNF-alpha). They were then subjected to nondenaturing polyacrylamide gel electrophoresis (PAGE). Binding of the native (slow) and activated (fast) forms of alpha(2)M to each cytokine was subjectively evaluated with autoradiography. Equine alpha(2)M bound I-125-TGF-beta 1. However, poor or no binding was observed between alpha(2)M and either of I-125-rhTNF-alpha or I-125-IL-1 beta. Synovial fluid was then obtained from 6 normal horses, 6 horses with septic arthritis, and 6 horses with degenerative joint disease. Untreated and methylamine-reacted samples were quantitatively examined for binding with I-125-TGF-beta 1, using the autoradiographic techniques described above and densitometry. Native and activated alpha(2)M were also quantified by densitometry of PAGE gels. Native alpha(2)M was significantly elevated in septic arthritis (6.4% to 29.5% of total protein detected) and degenerative joint disease (2.8% to 12.3%), compared to normal joints (0.9% to 4.2%). Activated alpha(2)M, however, was not detected in untreated synovial fluid samples. In all plasma and joint fluid samples, whether untreated or reacted with methylamine, I-125-TGF-beta 1 bound predominantly to alpha(2)M, and preferentially to the activated form of alpha(2)M. In synovial fluid, the amount of I-125-TGF-beta 1 binding was proportional to the quantity of alpha(2)M present. These results indicate that: 1) equine alpha(2)M binds TGF-beta 1; 2) the native form of alpha(2)M is present in both equine plasma and synovial fluid, and 3) alpha(2)M is a major binding protein for TGF-beta 1 in equine synovial fluid. Therefore, alpha(2)M may play a role in regulating this mediator of inflammation in equine joints.

The immune responsiveness of xenogeneic PBL engrafted into SCID mice was investigated using the bovine PBL-reconstituted SCID mouse model system (PBL-SCID-bo). Bovine PBL-reconstitution and B-cell activity were monitored by bovine serum Ig production. Bovine T-cell function was demonstrated by an antigen-specific immune response to bovine transplantation antigens provided by bovine skin allografts. Bovine allograft rejection was clearly evident in > 65% PBL-SCID-bo that received a bovine PBL inoculum either 30 days after bovine skin grafting, or 7–52 days before bovine skin grafting. Bovine allograft rejection was confirmed via histological examination and was characterized primarily by a band of infiltrating bovine lymphocytes at the periphery of the graft and tissue necrosis. A secondary immune response could be elicited if bovine cells in the PBL inoculum were presensitized to Ag from the bovine skin allograft donor. This study is the first to show that bovine cells engrafted in SCID mice after ip injection of bovine PBL retain some aspects of immune competency. These results confirm the value of the xenogeneic PBL-reconstituted SCID mouse model in the study of primary immunity.

The isotope dilution technique of [6-3H]glucose, [U-14C]lactate and [l-14C]propionate was used to evaluate the effect of dietary chromium (Cr) supplementation on whole-body kinetics of glucose, lactate, and propionate in rams. Rams were fed a high grain diet at 2% of body weight with or without 0.5 ppm of supplemental Cr from chelated Cr for the initial 14 days, and then intake was increased to 2.5% at body weight for the last 9 days. Weight gain was enhanced (P < 0.01) with Cr supplementation. Plasma concentrations of glucose, lactate, and propionate were not influenced by Cr supplementation. Turnover rates of glucose and lactate, and their interconversion were also not influenced. Propionate turnover rate tended to increase (P = 0.11) and the conversion of propionate to glucose increased (P < 0.05) with Cr supplementation, leading the increased proportional contribution of propionate to glucose turnover rate (P < 0.05). Chromium supplementation may influence the contribution of each glucogenic substrate for glucose production in rams fed a high grain diet.

The electrophoretic position and behavior of the native and activated forms of equine plasma alpha-2-macroglobulin (alpha 2M) were characterized and compared to human alpha 2M by nondenaturing polyacrylamide-gel electrophoresis (PAGE). Plasma alpha 2M was also compared between 6 normal horses and 6 horses with clinical signs of colic and endotoxemia due to volvulus or enteritis. Native and activated forms of alpha 2M were quantified by PAGE and densitometry. Binding of radio-labeled recombinant human tumour necrosis factor-alpha (125I-rhTNF-alpha) to native and activated forms of equine alpha 2M was also evaluated by autoradiography and densitometry of PAGE. Equine plasma alpha 2M migrated as a single band at a position equivalent to native human alpha 2M. Methylamine-reacted equine plasma samples resulted in faster migration of alpha 2M in a similar position to activated human alpha 2M. However, in methylamine-reacted equine plasma, an intermediate alpha 2M band was consistently present between the bands corresponding to native and activated alpha 2M. Amounts of plasma alpha 2M were similar in normal and endotoxemic horses, and remained in the electrophoretically slow or unreacted native form. The vast majority of 125I-rHuTNF-alpha did not bind to alpha 2M or other equine plasma proteins. 125I-rHuTNF-alpha bound weakly to both native and fast methylamine-reacted equine forms of alpha 2M, although binding was better to the activated form. This study indicates that: (1) equine plasma alpha 2M behaves similarly to human alpha 2M on PAGE, (2) plasma alpha 2M of horses can be activated to electrophoretically fast forms, but it is neither activated nor depleted during endotoxemia, and (3) the binding interactions between equine alpha 2M and TNF-alpha are too low to implicate equine alpha 2M as a regulator of TNF-alpha during endotoxemia in horses.

A simple method of catheterizing an artery which supplies the ipsilateral side of the mammary gland of a high-producing dairy cow is described. The preparation allows infusion of the mammary gland with nutrients or endocrine factors locally, but does not require general anaesthesia or deep surgical dissection. Following a preliminary experiment involving infusion of acetate, the catheters were removed and the cows returned to the milking herd. Key words: Cow, mammary gland, arterial infusion, acetate