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Abstract
Objective—To optimize the isolation and culture of mesenchymal stem cells (MSCs) from umbilical-cord blood (UCB), identify variables that predicted successful MSC isolation, and determine whether shipping, processing, and cryopreservation altered MSC viability, recovery rates, and expansion kinetics.
Sample Population—UCB samples from 79 Thoroughbred and Quarter Horse mares.
Procedures—UCB samples were processed to reduce volume and remove RBCs. Nucleated cells (NCs) were cryopreserved or grown in various culture conditions to optimize MSC monolayer expansion and proliferation. Donor and UCB-sample factors were analyzed to determine their influence on the success of MSC isolation and monolayer expansion.
Results—MSCs capable of multilineage in vitro differentiation were expanded from > 80% of UCB samples. Automated UCB processing and temperature-controlled shipping facilitated sterile and standardized RBC reduction and NC enrichment from UCB samples. The number of NCs after UCB samples were processed was the sole variable that predicted successful MSC expansion. The UCB-derived MSCs and NCs were successfully cryopreserved and thawed with no decrease in cell recovery, viability, or MSC proliferation. The use of fibronectin-coated culture plates and reduction of incubator oxygen tension from 20% to 5% improved the MSC isolation rate. Some UCB-derived MSC clones proliferated for > 20 passages before senescence. Onset of senescence was associated with specific immunocytochemical changes.
Conclusions and Clinical Relevance—Equine UCB samples appeared to be a rich source of readily obtainable, highly proliferative MSCs that could be banked for therapeutic use.
Abstract
Objective—To evaluate the short-term cardiovascular effects of IV administration of dipyrone (metamizole) as an intraoperative analgesic during total IV anesthesia with propofol.
Animals—6 healthy female New Zealand White rabbits.
Procedures—Anesthesia was induced with propofol (4.0 to 8.0 mg/kg, IV) and maintained with the same drug (1.2 to 1.3 mg/kg/min, IV). After induction, 3 doses of dipyrone (65 mg/kg each) were administered IV at 25-minute intervals. Before and for 10 minutes after each dipyrone injection, the following vascular and hemodynamic variables were recorded at the left common carotid artery every minute after the first injection: vessel diameter; peak systolic, minimum diastolic, end-diastolic, and mean blood flow velocities; mean volumetric flow; resistance and pulsatility indices; mean arterial blood pressure (MAP); heart rate; arterial oxygen saturation (SpO 2); and end-tidal partial pressure of CO2 (PETCO 2). Echocardiography was performed after the second injection. The same variables were measured at the abdominal aorta (AA) after the third injection.
Results—Dipyrone injections caused a significant, transient decrease in the resistance index at the AA. Also detected were a minor decrease in pulsatility index at the left common carotid artery and a minor increase in end-diastolic blood flow velocity at the AA. The MAP, heart rate, SpO 2, and PETCO 2 did not significantly change after injections. A comparison of HR and MAP after the first and third bolus injections revealed only minor changes.
Conclusions and Clinical Relevance—Dipyrone used with propofol anesthesia in rabbits appeared not to significantly impair cardiovascular and hemodynamic function.
Abstract
Objective—To determine plasma osmolality in healthy adult Hispaniolan Amazon parrots (Amazona ventralis) and validate osmolality equations in these parrots.
Animals—20 healthy adult Hispaniolan Amazon parrots.
Procedures—A blood sample (0.5 mL) was collected from the right jugular vein of each parrot and placed into a lithium heparin microtainer tube. Samples were centrifuged, and plasma was harvested and frozen at −30°C. Samples were thawed, and plasma osmolality was measured in duplicate with a freezing-point depression osmometer. The mean value was calculated for the 2 osmolality measurements.
Results—Plasma osmolality values were normally distributed, with a mean ± SD of 326.0 ± 6.878 mOsm/kg. The equations (2 × [Na+ + K+]) + (glucose/18), which resulted in bias of 2.3333 mOsm/kg and limits of agreement of −7.0940 to 11.7606 mOsm/kg, and (2 × [Na+ + K+]) + (uric acid concentration/16.8) + (glucose concentration/18), which resulted in bias of 5.8117 mOsm/kg and limits of agreement of −14.6640 to 3.0406 mOsm/kg, yielded calculated values that were in good agreement with the measured osmolality.
Conclusions and Clinical Relevance—IV administration of large amounts of hypotonic fluids can have catastrophic consequences. Osmolality of the plasma from parrots in this study was significantly higher than that of commercially available prepackaged fluids. Therefore, such fluids should be used with caution in Hispaniolan Amazon parrots as well as other psittacines. Additional studies are needed to determine whether the estimation of osmolality has the same clinical value in psittacines as it does in other animals.
Abstract
Objective—To compare effects of corn oil or a 7-carbon fat (triheptanoin) on acylcarnitine, lipid, and carbohydrate metabolism in plasma or muscle of exercising horses.
Animals—8 Thoroughbred geldings.
Procedures—Horses received isocaloric diets containing 650 mL of oil (triheptanoin or corn oil)/d for 18 or 25 days in a crossover design with a 26-day washout period. On day 17 or 24 of each feeding period, the respective oil (217 mL) was nasogastrically administered; 120 minutes later, horses performed a 90-minute submaximal exercise test (SET). Blood and muscle samples were obtained before oil administration and immediately before (blood only), during (blood only), immediately after, and 24 hours after SETs.
Results—Compared with values before oil administration, triheptanoin administration increased plasma insulin and C7:0-, C5:0- and C3:0-acylcarnitine concentrations, whereas corn oil administration increased plasma NEFA concentrations. During SETs, plasma C7:0-, C5:0-, and C3:0-acylcarnitine concentrations were higher when triheptanoin, rather than corn oil, was administered to horses. Plasma glucose, NEFA, and C2:0-, C18:1-, and C18:2-acylcarnitine concentrations increased during SETs similarly for both oils. Respiratory quotient and muscle lactate, citrate, malate, glycogen, and ATP concentrations changed similarly from before to after SETs for both oils. Compared with muscle concentrations immediately after SETs, those for glucose-6-phosphate and citrate 24 hours after SETs were lower and for glycogen were similar to values before SETs.
Conclusions and Clinical Relevance—Fatigue was not associated with depletion of citric acid cycle intermediates for either oil. Triheptanoin induced a significantly higher insulin secretion and did not appear to enhance muscle glycogen repletion.
Abstract
Objective—To characterize effects of IV administration of pirfenidone on clinical, biochemical, and hematologic variables and circulating tumor necrosis factor (TNF)-α concentrations in horses after infusion of a low dose of endotoxin.
Animals—18 healthy adult horses.
Procedures—Horses were randomly assigned to 3 groups (n = 6 horses/group) and administered an IV infusion of 30 ng of endotoxin/kg or saline (0.9% NaCl) solution during a 30-minute period. Lipopolysaccharide-pirfenidone horses received endotoxin followed by pirfenidone (loading dose of 11.6 mg/kg and then constant rate infusion [CRI] at 9.9 mg/kg/h for 3 hours). Lipopolysaccharide-saline horses received endotoxin followed by infusion (loading dose and CRI for 3 hours) of saline solution. Saline-pirfenidone horses received saline solution followed by pirfenidone (loading dose and then CRI for 3 hours). Physical examination variables were recorded and blood samples collected at predetermined intervals throughout the 24-hour study period. Blood samples were used for CBCs, biochemical analyses, and determinations of TNF-α concentrations.
Results—IV infusion of pirfenidone after administration of a low dose of endotoxin failed to attenuate the clinical, clinicopathologic, or cytokine alterations that developed secondary to endotoxin exposure. Intravenous infusion of pirfenidone after administration of saline solution induced mild transient clinical signs, but associated clinicopathologic changes were not detected.
Conclusions and Clinical Relevance—IV administration of pirfenidone was tolerated with only mild transient clinical adverse effects during infusion. However, administration of pirfenidone did not protect horses from the systemic effects of experimentally induced endotoxemia. Further studies of related, but more potent, drugs may be warranted.
Abstract
Objective—To develop an equation expressing urine pH in terms of independent variables, derive an equation relating urine pH to net acid excretion (NAE), and apply this new knowledge to determine the role that monitoring urine pH should play when diets with low cationanion difference are fed to dairy cattle.
Animals—11 Holstein-Friesian cows.
Procedures—A physicochemical strong ion approach was used to develop a general electroneutrality equation for urine that involved urine pH and strong ion difference (SID [difference between strong cation and strong anion concentrations]), PCO 2, the concentration of ammonium ([NH4 +]) and phosphate ([PO4]), and 3 constants. The general electroneutrality equation was simplified for use in bovine urine and applied to 321 data points from 11 cows fed different diets.
Results—Urine pH was dependent on 4 independent variables (urine SID, [NH4 +], PCO 2, and [PO4]) and 3 constants. The simplified electroneutrality equation for bovine urine was pH ≈ {pK1′ − log10(S PCO 2)} + log10([K+] + [Na+] + [Mg2+] + [Ca2+] + [NH4 +] − [Cl−] − [SO4 2−]). The relationship between urine pH and NAE (in mEq/L) for cattle fed different diets was pH = 6.12 + log10(−NAE + [NH4 +] + 2.6).
Conclusions and Clinical Relevance—A change in urine SID, [NH4 +], PCO 2, or [PO4] independently and directly led to a change in urine pH. Urinary [K+] had the greatest effect on urine pH in cattle, with high urine [K+] resulting in alkaline urine and low urine [K+] resulting in acidic urine. Urine pH provided an accurate assessment of NAE in cattle when pH was > 6.3.
Abstract
Objective—To measure epithelial cell percentages and somatic cell counts (SCCs) in milk and determine whether isoflupredone acetate reduces mammary gland epithelial cell sloughing in cows with acute endotoxin-induced mastitis.
Animals—13 lactating Holstein cows.
Procedures—Determination of SCC and flow cytometric analysis of cytokeratin-positive (epithelial) cells in milk were performed before and 12 hours after induction of mastitis via intramammary administration of bacterial endotoxin in 8 cows and at the same time points in 5 cows without mastitis. Endotoxin-treated cows received isoflupredone acetate (20 mg) or saline (0.9% NaCl) solution (n = 4/group) IV after signs of mastitis developed.
Results—At the 12-hour time point, mean ± SD percentage of epithelial cells in milk increased from 2.74 ± 1.93% to 42.11 ± 36.21% and decreased from 5.73 ± 4.52% to 5.31 ± 1.93% in milk from cows with and without mastitis, respectively. Median (range) SCC in milk increased from 195,000 cells/mL (17,000 to 442,000 cells/mL) to 5,437,500 cells/mL (69,000 to 11,036,000 cells/mL) and from 19,000 cells/mL (9,000 to 125,000 cells/mL) to 51,000 cells/mL (10,000 to 835,000 cells/mL) in cows with and without mastitis, respectively. Changes in these variables were significantly greater in mastitis-affected cows. Administration of isoflupredone acetate did not affect epithelial cell percentage or SCC in milk.
Conclusions and Clinical Relevance—During the early phase of endotoxin-induced mastitis in dairy cows, large numbers of epithelial cells were sloughed into the milk. Epithelial cell damage likely precedes an influx of immune cells into affected mammary glands and may contribute to breakdown of the blood-milk barrier.
Abstract
Objective—To develop a protocol to induce and maintain gastric ulceration in horses and to determine whether gastric ulceration affects physiologic indices of performance during high-speed treadmill exercise.
Animals—20 healthy Thoroughbreds.
Procedures—Each horse was acclimatized to treadmill exercise during a 2-week period. Subsequently, baseline data were collected (day 0) and each horse began an incrementally increasing exercise training program (days 1 through 56). Beginning on day 14, horses were administered omeprazole (4 mg/kg, PO, q 24 h until day 56) or no drug (10 horses/group) and underwent alternating 24-hour periods of feeding and feed withholding for 10 days to induce gastric ulceration. Extent of gastric ulceration was assessed weekly thereafter via gastroscopy. Physiologic indices of performance were measured at days 0 and 56. Gastric ulceration and exercise performance indices were compared within and between groups.
Results—In untreated horses, gastric ulcers were induced and maintained through day 56. Gastric ulcer formation was prevented in omeprazole-treated horses. There were significant interactions between time (pre- and post-training data) and treatment (nonulcer and ulcer groups) for mass-specific maximal O2 consumption (
O
2max/Mb) and mass-specific maximal CO2 production (
CO
2max/Mb). Post hoc analysis revealed a difference between groups for
O
2max/Mb at day 56. Within-group differences for
O
2max/Mb and
CO
2max/Mb were detected for omeprazole-treated horses, but not for the horses with ulcers.
Conclusions and Clinical Relevance—In horses, gastric ulcers were induced and maintained by use of alternating periods of feeding and feed withholding in association with treadmill exercise (simulated racetrack training). Gastric ulcers adversely affected physiologic indices of performance in horses.
Abstract
Objective—To establish an ex vivo model of blood perfusion in the distal portion of isolated equine forelimbs that closely represents the in vivo situation in the laminar tissue of the hoof.
Sample Population—18 forelimbs collected from 9 healthy adult horses following slaughter at a licensed abattoir.
Procedures—The distal portion of isolated equine forelimbs from 9 horses were perfused under physiologic conditions over a period of 6, 8, and 10 hours with autologous blood. To determine cell viability in perfused tissues, indicators for metabolism (lactate generation and glucose and oxygen consumption) as well as indicators for cell damage (potassium concentration and lactate dehydrogenase activity) were examined at 1-hour intervals from samples of the perfusate. Weight gain in the forelimb was used to determine the edema index. After perfusion, light and electron microscopic examinations of laminar tissue specimens were performed.
Results—During hemoperfusion of the isolated forelimbs, mean ± SD glucose consumption was 197.4 ± 65.1 mg/h, lactate generation was 1.84 ± 0.79 mmol/h, and oxygen consumption was 6.4 × 10−6 ± 8.9 × 10−5 mL·g−1·min−1. Neither an efflux of potassium into the perfusate nor a relevant increase of the lactate dehydrogenase activity was detected, indicating low amounts of cellular damage in the perfused tissues. Weight gain of forelimbs was 1.02 ± 0.95%. Histologic and ultrastructural appearance of the laminar tissue revealed no signs of tissue damage.
Conclusions and Clinical Relevance—Isolated equine limbs were perfused under physiologic conditions over a period of ≤ 10 hours without structural damage to the laminar tissue.
Abstract
Objective—To evaluate the effect of parenteral administration of ivermectin and erythromycin on abomasal emptying rate in suckling calves.
Animals—6 male Holstein-Friesian calves < 15 days old.
Procedures—In a crossover study, calves were administered each of 3 treatments (control treatment, 2 mL of saline [0.9% NaCl] solution, IM; erythromycin, 8.8 mg/kg, IM; and ivermectin, 200 μg/kg, IV). Thirty minutes later, calves were bottle-fed 2 L of fresh cow's milk containing acetaminophen (50 mg/kg). Blood samples were collected from a jugular vein at various periods after suckling of milk. Abomasal emptying rate was assessed by use of the time to pharmacokinetically determined maximal plasma acetaminophen concentration.
Results—Administration of erythromycin and ivermectin caused a significant increase in abomasal emptying rate, compared with results for the control treatment, as determined on the basis of time to maximal plasma acetaminophen concentration.
Conclusions and Clinical Relevance—Parenteral administration of erythromycin and ivermectin increased the abomasal emptying rate. The macrolide erythromycin can be an effective prokinetic agent in calves and other animals. Ivermectin is classified as a macrolide but has a number of structural differences from erythromycin. The clinical importance of a slight increase in abomasal emptying rate after IV administration of ivermectin remains to be determined because ivermectin is only labeled for SC, oral, and topical administration.
