In vitro comparison of the effects of two forms of hydroxyethyl starch solutions on platelet function in dogs

OBJECTIVE: To evaluate the effect of 2 hydroxyethyl starch (HES) preparations (ie, HES solution with a molecular weight of 600 kd and a degree of substitution of 0.7 [HES 600/0.7] and a calcium-containing polyionic HES solution with a molecular weight of 670 kd and a degree of substitution of 0.75 [HES 670/0.75]) on canine platelet function. SAMPLE POPULATION: Blood samples from 10 healthy adult dogs. PROCEDURES: Dilution of citrated whole blood was performed with saline (0.9% NaCl) solution, HES 600/0.7, and HES 670/0.75 at ratios of 1:9 (ie, 1 part saline solution or colloid to 9 parts whole blood) and 1:3. Measurements of time to platelet plug formation in a capillary tube (ie, closure time) were made by use of a bench-top platelet function analyzer with collagen and ADP platelet agonists. RESULTS: Mean baseline closure time was 68.0 +/- 15.3 seconds. A 1:3 dilution of whole blood with saline solution, HES 600/0.7, and HES 670/0.75 resulted in mean closure times of 85.8 +/- 15.7 seconds, 100.6 +/- 18.6 seconds, and 101.6 +/- 16.2 seconds, respectively. Closure time following 1:3 dilution of whole blood with saline solution was significantly different from baseline and from 1:9 dilution with saline solution. Closure time following 1:3 dilution of whole blood with HES 670/0.75 was significantly different from baseline, 1:3 and 1:9 dilutions with saline solution, and 1:9 dilutions with HES 600/0.7 or HES 670/0.75. CONCLUSIONS AND CLINICAL RELEVANCE: Saline solution, HES 600/0.7, and HES 670/0.75 affect canine platelet function by prolonging closure times; HES solutions prolonged closure time to a greater extent than saline solution.     

Stomatocytosis in 7 related Standard Schnauzers

BACKGROUND: Hereditary canine stomatocytosis has been described in purebred Alaskan Malamutes, Drentse Patrijshonds, and Miniature Schnauzers. In humans, hereditary stomatocytosis is a heterogeneous group of congenital disorders characterized by the presence of stomatocytes in blood, increased osmotic fragility, and frequently, hemolytic anemia. OBJECTIVE: Our objective was to describe hematologic findings and RBC characteristics in 7 closely related Standard Schnauzers with stomatocytosis. METHODS: The following parameters were measured using an automated analyzer: HCT, RBC, hemoglobin (Hb) concentration, MCV, MCH, MCHC, red cell distribution width (RDW), WBC, platelet count, mean platelet volume (MPV), thrombocrit (PCT), and platelet distribution width (PDW). Differential leukocyte count, platelet estimate, reticulocyte count, and the percentage of stomatocytes in blood films were microscopically evaluated. An osmotic fragility test of RBCs and measurement of intracellular Na+, K+, and 2,3-diphosphoglycerate (2,3-DPG) concentrations were also performed. RESULTS: The affected dogs had macrocytosis (80.0 +/- 4.2 fL, reference interval 60-76 fL), decreased MCHC (29.3 +/- 0.8 g/dL, reference interval 32-39 g/dL), slightly increased RDW (17.3 +/- 0.4%, reference interval 12-16%), and an increased reticulocyte count (1.55 +/- 0.77%, reference interval <1%). The percentage of stomatocytes in blood films varied from 0.6 to 18.9% of all RBCs. Erythrocyte osmotic fragility and intracellular Na+ (138.1 +/- 3.2 mmol/L; controls 99 +/- 6.1 mmol/L), K+ (8.1 +/- 0.8 mmol/L; controls 6.1 +/- 0.5 mmol/L), and 2,3-DPG (21.9 +/- 2.0 micromol/g Hb; controls: 14.6 +/- 3.3 micromol/g Hb) concentrations were increased in dogs with stomatocytosis. CONCLUSIONS: Hematologic findings and the metabolic defects in RBCs in these Standard Schnauzers were consistent with a diagnosis of stomatocytosis. Parentage analysis suggests that stomatocytosis in Standard Schnauzers may have a hereditary component.     

Hemolytic potential of guaifenesin in cattle

The hemolytic effect on bovine red blood cells of 5%, 10%, and 15% guaifenesin solutions in 5% dextrose, 0.9% saline (NaCl), or distilled water was determined in vitro at 2 plasma concentrations (250 micrograms/ml, 500 micrograms/ml). A solution of 5% guaifenesin in a 5% dextrose solution or 5% guaifenesin in 0.9% saline produced minimal hemolysis in vitro. The amount of hemolysis of bovine red blood cells in vitro was related to the concentration of guaifenesin, diluent (5% dextrose, 0.9% NaCl, distilled water) and the plasma concentration of guaifenesin. In addition, plasma hemoglobin was determined in 4 adult dairy cows following the IV administration of 5% and 10% guaifenesin. These studies suggest that a solution of 5% guaifenesin in 5% dextrose is the most suitable solution for clinical use in cattle.     

Influence of transfusion technique on survival of autologous red blood cells in the dog

Objective – To determine the effect of 3 differing transfusion techniques on survival of autologous canine RBCs. Design – Prospective, blinded study. Setting – University Teaching Hospital. Animals – Nine healthy dogs. Interventions – Three distinct preparations of RBCs, each representing ～1% of red cell mass, were generated for each dog by biotinylation of RBCs at varying biotin densities. Labeled cells were transfused using 3 techniques (gravity, volumetric pump, syringe pump). Serial determinations of red cell survival were carried out by flow‐cytometric analysis of RBCs collected at 7‐day intervals for 49 days. In vitro analysis of the effect of transfusion methods on RBC integrity and osmotic fragility were carried out in 7/9 dogs. Measurements and Main Results – RBCs administered via volumetric and syringe pumps exhibited a marked decrease in short‐term probability of survival compared with RBCs delivered by gravity flow. At 24 hours, only 4/8 and 1/7 dogs had surviving cell populations delivered by volumetric and syringe pump, respectively, compared with 8/8 dogs which had surviving cell populations delivered by gravity flow. Circulating half‐life of cells surviving at 24 hours after delivery by volumetric pump was not significantly different to that delivered by gravity flow. No significant effect on in vitro RBC integrity or osmotic fragility was detected in relation to transfusion technique. Conclusions – Delivery of autologous canine RBCs via mechanical delivery systems was associated with a high risk for early loss of transfused cells.     

Effect of hydroxyethyl starch solution in normal horses and horses with colic or acute colitis

Hydroxyethyl starch (HES) solution is an effective colloidal infusion solution in humans for treatment of hypovolaemic shock, but it has not been compared with fluids currently available for use in horses. On the basis of plasma-expanding effect of HES in normal horses, a 10% medium-molecular 200/0.5 solution of HES was subsequently tested in hypovolaemic horses. Six normal horses were given five protocols of a single infusion of HES at varying dosage rates (5, 10, 15 ml HES/kg), as well as isotonic saline (15 ml/kg) and hypertonic saline (4 ml/kg b.w.). Dehydrated horses suffering from acute colitis or those which had been treated surgically for ileus of the small or large intestine were given an i.v. infusion of 10 ml HES/kg in combination with 10 ml saline/kg. Clinical data and blood samples for testing were taken before the infusion, and then 10 min, 1 h, 2, 4, 6, 8, 10, 12 and 24 h after infusion (a.i.). A significant decrease in haematocrit was observed in protocol 1-5 for a period of up to 4, 4, 10 h, 10 min and up to 10 min; in group of colitis, during the entire 24-h testing period, and in groups of ileus of small intestine and of large intestine, up to 4 and 10 h a.i. HES decreases better and longer-lasting haematocrit and total protein than either isotonic or hypertonic saline. Half-life of HES increases due to higher dosage (5.83, 7.63 and 11.48 h) and distribution is exclusively intravascular. In normal horses of protocol 1-3 using HES aPTT, sodium and potassium were within the physiological range. Serum amylase activity is increased in horses using HES. On the basis of this clinical study, the decreasing effect of urea and creatinine in colic patients after surgery and fewer instances of postoperative ileus a dosage of 10 ml HES/kg could be recommended.     

Comparison of glycerol, lactamide, acetamide and dimethylsulfoxide as cryoprotectants of Japanese white rabbit spermatozoa

The rabbit is considered to be a valuable laboratory animal. We compared glycerol, lactamide, acetamide, and dimethylsulfoxide (DMSO) as cryoprotectants in egg-yolk diluent of ejaculated Japanese white rabbit spermatozoa for improvement of sperm cryopreservation methods. Rabbit semen was frozen with 1.0 M glycerol, lactamide, acetamide, or DMSO in plastic straws. Forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rate of forward progressive motile spermatozoa in lactamide (37.8 +/- 3.0%) was significantly (P<0.05) higher than in glycerol (17.0 +/- 3.3%). In addition, the rates of sperm plasma membrane integrity in lactamide and acetamide (35.9 +/- 3.3% and 30.2 +/- 3.0%, respectively) were significantly (P<0.05) higher than in glycerol (17.0 +/- 2.6%). The results indicate that 1.0 M lactamide and acetamide have higher cryoprotective effects than 1.0 M glycerol for cryopreservation of Japanese white rabbit spermatozoa.     

Abnormality of osmotic fragility and morphological disorder of bovine erythrocytes infected with Theileria sergenti

The osmotic fragility and the surface structure of erythrocytes obtained from 3 calves infected with Theileria sergenti and from 3 phlebotomized ones were compared. As the parasitemia progressed, the osmotic fragility of the erythrocytes significantly increased in the infected calves. Particularly the hemolysis ratio in the isotonic area (21.5-94.1%) obviously increased. On the other hand, the percentage of parasitized cells in the erythrocytes did not show so much high values (16.1-21.3%). Similar phenomenon was found in each different percentage of erythrocytes suspension which was separated from density gradient centrifugation. No significant difference in the serum osmotic pressure between the infected calves and the phlebotomized calves was found. By scanning microscopy, the erythrocytes of infected calves, which were collected at the crisis period of parasitemia, were almost completely deformed and showed echinocyte form. Moreover, the appearance ratio of echinocyte form in the erythrocytes population was superior to the percentage of parasitized erythrocytes. Similar membranous alterations were also observed in the erythrocytes of grazing cattle in the crisis period of the theileriosis. It was proven that abnormality of osmotic fragility and morphological disorders of erythrocytes occurred not only in parasitized erythrocytes but also in non-parasitized ones in T. sergenti parasitemia.     

Effects of epidural administration of morphine and buprenorphine on the minimum alveolar concentration of isoflurane in cats

OBJECTIVE: To determine effects of epidural administration of morphine and buprenorphine on the minimum alveolar concentration of isoflurane in cats. Animals-6 healthy adult domestic shorthair cats. PROCEDURES: Cats were anesthetized with isoflurane in oxygen. Morphine (100 microg/kg diluted with saline [0.9% NaCl] solution to a volume of 0.3 mL/kg), buprenorphine (12.5 microg/kg diluted with saline solution to a volume of 0.3 mL/kg), or saline solution (0.3 mL/kg) was administered into the epidural space according to a Latin square design. The minimum alveolar concentration (MAC) of isoflurane was measured in triplicate by use of the tail clamp technique. At least 1 week was allowed between successive experiments. RESULTS: The MAC of isoflurane was 2.00 +/- 0.18%, 2.13 +/- 0.11%, and 2.03 +/- 0.09% in the morphine, buprenorphine, and saline solution groups, respectively. No significant difference in MAC was detected among treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: A significant effect of epidural administration of morphine or buprenorphine on the MAC of isoflurane in cats could not be detected. Further studies are needed to establish whether epidural opioid administration has other benefits when administered as a component of general anesthesia in cats.     

Evaluation of Citrate-Phosphate- Dextrose-Adenine as a Storage Medium for Packed Canine Erythrocytes

Packed canine red blood cells (RBCs) stored in the anticoagulant-preservative solution citrate-phosphate-dextrose-adenine (CPDA-1) were studied at 1, 10, 20, 30, and 40 days. The extracellular concentrations of potassium and sodium, erythrocyte mean corpuscular volume, and osmotic fragility increased during storage (P less than 0.05). There was a decrease in the pH, plasma concentration of glucose, and erythrocyte concentrations of 2,3-diphosphoglycerate (2,3-DPG) and adenosine-5'-triphosphate (P less than 0.05). Erythrocyte 2,3-DPG concentration decreased by 54% within the first 24 hours of storage (P less than 0.001). Posttransfusion viability (PTV) decreased from 90% on day 1 to 46% on day 40 (P less than 0.05). The PTV of the RBCs stored for 10 and 20 days complied with the Food and Drug Administration (FDA) standard. Although there are marked biochemical and hematologic changes in stored packed red blood cells (pRBCs), 20-day-old units may be expected to be of acceptable quality. The sharp decrease in 2,3-DPG concentration suggests a reduction in oxygen carrying capacity in erythrocytes stored as pRBCs. Hyperkalemia occurs during storage of pRBCs and does not appear to be associated with high intraerythrocytic potassium concentrations.     

Effect of intravenous infusion of a 7.2% hypertonic saline solution on serum electrolytes and osmotic pressure in healthy beagles.

The effect of an intravenous (i.v.) infusion of hypertonic saline solution (HSS; 7.2%, 2,400 mOsmol/kg.H2O) was evaluated by serum electrolyte concentrations and osmotic pressure in the anesthetized beagles. Sixteen beagles were assigned to 3 experimental groups (2.5, 5 or 15 ml/kg of HSS i.v. infusion) or a control group (5 ml/kg of isotonic saline solution (ISS) i.v. infusion) and were monitored for 120 min after the initiation of fluid infusion. The relative plasma volume (rPV) in the 5 ml/kg and 15 ml/kg HSS groups progressively expanded to 143.1 +/- 7.4% at 3 min and 156.4 +/- 5.9% at 5 min after the initiation of the fluid infusion, respectively. Significant increases were not produced by ISS and 2.5 ml/kg HSS infusion. The serum sodium and chloride concentrations in the ISS group were not altered. The 5 ml/kg HSS infusion induced transient high osmotic and sodium levels, and the serum sodium concentration remained under the 160 mM/l after the completion of the HSS infusion. However, the 15 ml/kg HSS infusion induced a constant high osmotic level (340.5-352.8 mOsmol/kg.H2O) and hypernatremia (161.4-174.5 mM/l) from 10 to 90 min after the initiation of the fluid infusion. The 15 ml/kg HSS infusion induced significant decreases in the partial pressure of oxygen (PaO2), reaching 63.7 +/- 8.0 mmHg at 120 min after the initiation of the fluid infusion compared with an immediately before fluid infusion value. On the basis of these findings, 5 ml/kg HSS infusion can be safely administered to healthy beagles for expanding the plasma volume without inducing hypernatremia. A 5 ml/kg HSS infusion is thus recommended for the initial field resuscitation of dogs.     

Glycerol hyperhydration in resting horses

SUMMARY: To determine whether administration of glycerol-containing solutions induces a state of transient hyperhydration in resting euhydrated horses, changes in plasma and urine constituents were measured in four horses for 1 h before and 5 h after nasogastric administration of each of four treatments (Experiment 1). Treatments were applied in a randomized fashion and included: (1) 1.0 g.kg(-)(1)glycerol in 8 L of water (G); (2) 8 L of water (W); (3) 8 L of 0.9% NaCl solution (S); and (4) 1.0 g.kg(-)(1)glycerol in 8 L of 0.9% NaCl solution (GS). In a subsequent study, voluntary water intake was measured hourly for 5 h after nasogastric administration of each treatment (Experiment 2). All treatments produced mild plasma volume expansion ranging from 3.2 to 5.8% in Experiment 1. Administration of glycerol containing solutions increased serum glycerol concentration approximately 100-fold and plasma osmolality (P(osm)) by approximately 10 mOsm/kg and resulted in a tendency towards increased renal water conservation despite increased osmole excretion. In contrast, W treatment decreased plasma and urine osmolality and was accompanied by increased urine production and decreased renal water conservation. Plasma and urine osmolality, as well as renal osmole and water excretion, were unchanged after S administration. In Experiment 2, horses treated with GS voluntarily drank an additional 5.2 +/- 0.9 L of water during the initial hour following nasogastric administration of 8 L of solution. Voluntary water intake with the other treatments was less than 1.0 L for the entire 5 h observation period. Collectively, the results of both experiments suggest that administration of glycerol in saline would produce transient hyperhydration in resting euhydrated horses by enhancing renal water conservation and stimulating voluntary water intake.     

The effect of polyethylene glycol-200 on metabolic acidosis induced by chloralose anaesthesia

Chloralose may be used in a 10% solution as an anaesthetic in dogs. The solubility of chloralose was found to be much higher in polyethylene glycol-200 (PEG-200) than in either warm (body temperature) or cold saline (0.9% NaCl). The intravenous (i.v.) administration of chloralose in warm saline solution induced acidosis as a result of the increase in the level of metabolic acids. The acidity generated by chloralose in almost neutral saline was probably the result of increase in the base deficit in the animal. The infusion of PEG-200 (almost neutral) significantly reduced the base deficit without disturbing the PaO2 or PaCO2 in the arterial blood. The base deficit value was significantly lower after administration of chloralose solution in PEG-200 (almost neutral) than after administration in saline. The use of PEG-200 as a solvent for chloralose was advantageous in two ways. Firstly, it prevented the production of acids in anaesthetic solution and neutralized the blood metabolic acids generated by chloralose administration in saline. Secondly, the solubility of chloralose (10% w/v solution) in PEG-200 was very much higher than in warm or cold saline.     

Apparent pseudohyperkalemia in a Chinese Shar Pei dog

Whole blood in a serum clot tube and EDTA-anticoagulated samples from an 8-year-old spayed female Chinese Shar Pei dog were submitted by an external clinic to the diagnostic laboratory at Atlantic Veterinary College for routine biochemical and hematologic analysis prior to entropion surgery. Laboratory abnormalities included mild hyperkalemia (6.3 mmol/L, reference interval 3.6-6.0 mmol/L), mild normocytic, hypochromic, nonregenerative anemia (HCT 0.31 L/L, reference interval 0.37-0.55 L/L; MCHC 290 g/L, reference interval 320-360 g/L), and increased red cell distribution width (RDW; 26.2%, reference interval 11-14%). A small subpopulation of macrocytic, slightly hypochromic erythrocytes was noted on Wright's-Giemsa-stained blood smears. Biochemical and hematologic data obtained from this patient over the previous 7.5 years indicated that serum (and in 1 case, heparinized plasma) potassium concentration was increased (range, 6.3-10.9 mmol/L) in 5 of 8 samples (HCT ranged from 0.31-0.43 L/L, Hgb 91-124 g/L, MCHC 280-312 g/L, and RDW 18.2-26.9%). Clinical signs suggestive of hyperkalemia were not observed at any time, suggesting pseudohyperkalemia as the cause of the increased potassium concentrations. An erythrocyte lysate prepared from a heparinized blood sample had a high potassium concentration (16.8 mmol/L) compared with that of a clinically healthy, non-Shiba control dog (6.7 mmol/L). An osmotic fragility test of the patient's erythrocytes showed 50% hemolysis at 0.57% NaCl, compared with 0.48% NaCl for the control dog, indicating increased fragility. On scanning electron microscopy, a small subpopulation of erythrocytes were large, flattened, and had a tendency to fold. These findings supported the provisional diagnosis of pseudohyperkalemia due to increased intracellular RBC potassium concentration. High-potassium erythrocytes have been reported in Akitas, Shibas, Jindos, other East Asian dog breeds, and occasionally, in mixed-breed dogs. Pseudohyperkalemia should also be considered when an otherwise unexplained elevation in serum or plasma potassium concentration is observed in Chinese Shar Pei dogs, and may be accompanied by increased RDW, low MCHC, and increased osmotic fragility with or without mild anemia.     

The use of the rapid osmotic fragility test as an additional test to diagnose canine immune-mediated haemolytic anaemia

Background

Diagnosing canine immune-mediated haemolytic anaemia (IMHA) is often challenging because all currently available tests have their limitations. Dogs with IMHA often have an increased erythrocyte osmotic fragility (OF), a characteristic that is sometimes used in the diagnosis of IMHA. Since the classic osmotic fragility test (COFT) is time-consuming and requires specialized equipment, an easy and less labour-intensive rapid osmotic fragility test (ROFT) has been used in some countries, but its diagnostic value has not yet been investigated.This study aimed to evaluate erythrocyte osmotic fragility in dogs with and without IMHA, to compare results of the classic (COFT) and rapid (ROFT) test and to assess the value of the ROFT as diagnostic test for canine IMHA.Nineteen dogs with IMHA (group 1a), 21 anaemic dogs without IMHA (group 1b), 8 dogs with microcytosis (group 2), 13 hyperlipemic dogs (group 3), 10 dogs with lymphoma (group 4), 8 dogs with an infection (group 5) and 13 healthy dogs (group 6) were included.In all dogs, blood smear examination, in-saline auto-agglutination test, Coombs’ test, COFT and ROFT were performed. In the COFT, OF5, OF50 and OF90 were defined as the NaCl concentrations at which respectively 5, 50 and 90% of erythrocytes were haemolysed.

Results

Compared with healthy dogs, OF5 and OF50 were significantly higher in group 1a (P < 0.001) and OF5 was significantly higher in group 3 (P = 0.0266). The ROFT was positive in 17 dogs with IMHA, 10 hyperlipemic dogs, one anaemic dog without IMHA and one healthy dog.

Conclusions

Osmotic fragility was increased in the majority of dogs with IMHA and in dogs with hyperlipidemia, but not in dogs with microcytosis, lymphoma or an infection. Although more detailed information was obtained about the osmotic fragility by using the COFT, the COFT and ROFT gave similar results. The ROFT does not require specialized equipment, is rapid and easy to perform and can be used easily in daily practice. Although, the ROFT cannot replace other diagnostic tests, it may be a valuable additional tool to diagnose canine IMHA.     

Evaluation of a short-term saline diuresis protocol for the administration of cisplatin

A study was undertaken to determine the toxic effects of cisplatin, an antineoplastic agent, on canine kidneys and bone marrow when administered during a 6-hour saline diuresis. Cisplatin (70 mg/m2 of body surface) was administered IV to 6 healthy dogs over a 20-minute period after 0.9% NaCl solution (saline) was administered IV for 4 hours at a rate of 18.3 ml/kg/hr. After cisplatin injection, saline diuresis was continued at the same rate for 2 hours. Each dog vomited within 8 hours after the drug was administered. Clinical status, weight gain, and food consumption were normal throughout the 27-day study. All measures of renal function remained unchanged and were within normal limits for 27 days after the drug was administered. Nadirs in the daily neutrophil count were observed on days 6 (3,240 +/- 404/microliters) and 15 (1,196 +/- 275/microliters). There were no important gross or histologic abnormalities referable to cisplatin administration when the dogs were necropsied at the conclusion of the study (day 27). We concluded that cisplatin can be administered safely at a dosage of 70 mg/m2 of body surface, using a short-term diuresis protocol, and that the drug induces a nadir in the neutrophil count on days 6 and 15.     

Effect of medetomidine administration on bispectral index measurements in dogs during anesthesia with isoflurane

OBJECTIVE: To determine the relationship between bispectral index (BIS) and minimum alveolar concentration (MAC) multiples of isoflurane after IM injection of medetomidine or saline (0.9% NaCl) solution in anesthetized dogs. ANIMALS: 6 dogs. PROCEDURE: Each dog was anesthetized 3 times with isoflurane. First, the MAC of isoflurane for each dog was determined by use of the tail clamp method. Second, anesthetized dogs were randomly assigned to receive an IM injection of medetomidine (8 microg x kg(-1)) or an equal volume of isotonic saline (0.9% NaCl) solution 30 minutes prior to beginning BIS measurements. Last, anesthetized dogs received the remaining treatment (medetomidine or isotonic saline solution). Dogs were anesthetized at each of 4 MAC multiples of isoflurane. Ventilation was controlled and atracurium (0.2 mg/kg followed by 6 microg/kg/min as a continuous infusion, IV) administered. After a 20-minute equilibration period at each MAC multiple of isoflurane, BIS data were collected for 5 minutes and median values of BIS calculated. RESULTS: BIS significantly decreased with increasing MAC multiples of isoflurane over the range of 0.8 to 2.0 MAC. Mean (+/- SD) MAC of isoflurane was 1.3 +/- 0.2%. During isoflurane-saline anesthesia, mean BIS measurements at 0.8, 1.0, 1.5, and 2.0 MAC were 65 +/- 8, 60 +/- 7 52 +/- 3, and 31 +/- 28, respectively. During isoflurane-medetomidine anesthesia, mean BIS measurements at 0.8, 1.0, 1.5, and 2.0 MAC were 77 +/- 4, 53 +/- 7, 31 +/- 24, and 9 +/- 20, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: BIS monitoring in dogs anesthetized with isoflurane has a predictive value in regard to degree of CNS depression. During isoflurane anesthesia, our results support a MAC-reducing effect of medetomidine.     

Cryopreservation of Sheep Primordial Follicles

The aim of this study was to evaluate the efficiency of 1 M dimethylsulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PROH) and glycerol (GLY) to cryopreserve primordial follicles. The first evaluation was performed soon after cryopreservation and the second evaluation after 4 days of in vitro culture, using the cryoprotectants that allowed the higher results (higher follicular survival rate) after cryopreservation. The results after follicular isolation (control) and cryopreservation using 1 M DMSO, EG, PROH and GLY showed that the mean number (+/- SEM) of live follicles per millilitre was 3204 (100%) +/- 319.27, 2798 (87%) +/- 239.14, 2492 (78%) +/- 345.8, 448 (14%) +/- 46.3 and 208 (7%) +/- 75.26, respectively. Higher follicular survival was reported when DMSO and EG were used. Control follicles and follicles cryopreserved with these two cryoprotectants were cultured and the percentage of follicular survival was 55% (control), 42% (EG) and 34% (DMSO). Similar results were found between control and follicles cryopreserved with EG. In conclusion, 1 M EG is the most effective cryoprotectant to preserve primordial follicles isolated from ovaries of sheep.     

Conventional freezing of in vitro-produced and biopsied bovine blastocysts in the presence of a low concentration of glycerol and sucrose

The purpose of this study was to develop a practical cryopreservation method for in vitro-produced (IVP) and sex-predetermined bovine blastocysts that will be applicable to direct transfer of the post-thaw embryos. Blastocysts were harvested 7 days after IVF and allocated to either an intact or biopsy group. The cryoprotective solution contained 0.7 M glycerol and 0, 0.05 or 0.1 M sucrose. Slow cooling at a rate of -0.5 C/min was terminated at -25, -30, or -35 C, and rapid cooling in liquid nitrogen was followed. After one-step thawing and dilution, the IVP blastocysts were cultured for 3 days to assess their survival. The post-thaw survival rate of intact blastocysts after termination of slow cooling at -30 C in 0.7 M glycerol plus 0.1 M sucrose (96.2%) was significantly higher than that at -25 C in 0.7 M glycerol alone (44.4%). The post-thaw survival rate of biopsied bovine blastocysts after termination of slow cooling at -25 C in 0.7 M glycerol alone (53.8%) tended to be lower than that at -25 C in 0.7 M glycerol plus 0.05 M sucrose (91.3%) or -30 C in 0.7 M glycerol plus 0.1 M sucrose (92.3%). Thus, addition of a small amount of sucrose to 0.7 M glycerol cryoprotective solution shortened the process of slow cooling for both the intact and biopsied bovine embryos. Judged from the survival levels in vitro after thawing and one-step dilution of embryos (>80%), this is an improved method of cryopreservation for subsequent direct transfer of IVP and biopsied bovine blastocysts.     

Glycerol,Methyl‐Formamide and Dimethyl‐Formamide in Canine Semen Cryopreservation

The aim of the present work was to compare the efficiency of methyl‐formamide (MF), dimethyl‐formamide (DF) and glycerol (GL) as cryoprotectants in canine semen cryopreservation. For the experiment, pooled semen was submitted to one of the three cryoprotectants, with a final concentration of 3% in egg yolk–TRIS extender. Semen was subjectively evaluated for total and progressive motility, vigour and morphology. Sperm membrane functional integrity was assessed by hypo‐osmotic swelling test (HOST), and longevity was assessed using the thermoresistance test (TRT). Fresh semen showed normal physical and morphological characteristics. After thawing, differences were observed between semen frozen using GL and DF, regarding total and progressive motility and vigour (p < 0.05), but not between MF and GL or MF and DF. Means for total motility, progressive motility, vigour and morphologically normal spermatozoa were, respectively, 69.0 ± 5.4%, 61.0 ± 7.4%, 2.9 ± 0.5 and 57.1 ± 5.0% for GL; 59.0 ± 8.9%, 50.0 ± 10.0%, 2.5 ± 0.7 and 66.9 ± 7.7% for MF; and 44.0 ± 21.0%, 37.0 ± 19.8%, 2.1 ± 0.6 and 61.1 ± 5.5% for DF. On HOST, GL was superior (p < 0.05) to MF and DF (57.8 ± 12.4%, 35.8 ± 18.4% and 34.4 ± 9.4%, respectively). During the TRT, both GL and MF were superior to DF, with no differences between GL and MF. In conclusion, the use of MF as cryoprotectant showed results similar to GL, and can be considered as an alternative in canine semen cryopreservation. Further studies testing different concentrations of MF may improve its effects on cryopreservation of canine semen.     

Improved monospermic fertilization and subsequent blastocyst formation of bovine oocytes fertilized in vitro in a medium containing NaCl of decreased concentration

The objective of this study was to evaluate whether changes in NaCl concentration in a fertilization medium could improve normal fertilization and preimplantation development of bovine oocytes. In vitro matured bovine oocytes were inseminated with frozen-thawed semen for 18 hr in a Tyrode's medium with albumin, lactate and pyruvate (TALP), to which 114 (TALP-114), 96 (TALP-96) or 78 (TALP-78) mM NaCl was added. Presumptive zygotes were cultured for 192 hr in a modified TALP containing 90 mM NaCl, 1.5 mM glucose, 0.3% (w/v) BSA, minimal essential medium (MEM) essential and nonessential amino acids, and insulin-transferrin-selenium complex. Lower polyspermy rate was obtained by the insemination in TALP-96 (7.8 +/- 2.3%) than by the insemination in TALP-114 (25.6 +/- 1.4%), without decrease in male pronucleus (MPN) formation. Fertilization in TALP-78 also yielded decreased polyspermic fertilization (3.8 +/- 1.5%), but significant decrease in MPN formation was found (63.1 +/- 3.1%). In preimplantation development, more blastocysts developed from oocytes inseminated in TALP-96 (24.1 +/- 1.7%) than from oocytes inseminated in TALP-114 (16.8 +/- 1.4%). TALP-78, however, did not improve preimplantation development beyond the 8-cell stage compared with TALP-114. Mean cell number of blastocyst was higher when oocytes were fertilized in TALP-96 (137.0 +/- 4.5) than in TALP-114 (123.1 +/- 5.1) and in TALP-78 (102.3 +/- 4.5). These results demonstrate that insemination of bovine oocytes in a TALP with decreased NaCl concentration (96 mM) improves blastocyst formation and embryo viability. Decrease in NaCl concentration below 96 mM, however, may delay or inhibit MPN formation, and inhibits subsequent development in vitro.