Environmental survival of Haemophilus somnus and influence of secretions and excretions.

Environmental survival of the Haemophilus somnus virulent strain 43826 was examined by mixing it with bovine secretions and excretions and observing viability after storage at -70 degrees C, 3 degrees C, 23.5 degrees C and 37 degrees C at one day, five days, 12 days, 19 days and intermittently up to 75 days. Survival of the organism beyond 70 days occurred when it was mixed with cerebrospinal fluid, whole blood, blood plasma, vaginal mucus and milk and frozen at -70 degrees C. At 3 degrees C the organism in these fluids survived for five days or less. At 23.5 degrees C the organism survived beyond 70 days when mixed with whole blood and nasal mucus. The viability of H. somnus in urine at all temperatures was less than 24 hours and less than 15 minutes at 20 degrees C and 37 degrees C. Infective cerebrospinal fluid frozen alone in liquid nitrogen and with the addition of various cryopreservatives allowed the organism to survive and maintain virulence for at least 56 days. The implications of these studies to disease transmission and experimental studies is discussed.     

Factors affecting the survival of Streptococcus suis type 2

The survival of Streptococcus suis type 2 was assessed in experimentally inoculated faeces and dust stored at 0, 9 and 22 to 25 degrees C. The organism survived in faeces for 104 days at 0 degrees C, up to 10 days at 9 degrees C and up to eight days at 22 to 25 degrees C. It survived in dust for up to 54 days at 0 degrees C and up to 25 days at 9 degrees C but could not be isolated from dust stored at room temperature for 24 hours. The organism survived at 4 degrees C in nutrient medium for up to nine months but in distilled water for only one to two weeks. At 50 degrees C it survived in water or broth for up to two hours but at 60 degrees C it only survived for 10 minutes. The organism was rapidly inactivated by disinfectants and cleansers, commonly used on farms and in laboratories, at concentrations less than those recommended for use by the manufacturers.     

The effect of the method of adding ethylene glycol to turkey ejaculate on the quality of cryopreserved sperm

Ethylene glycol at concentrations of 8 + 8, 4 + 12 or 0 + 16% was added to the ejaculate of the White Broad-Breasted turkey-stags by the method of (ejaculate : diluent) 1 : (1 + 1), 1 : (1 + 1/4 + 1/4 + 2/4) or 1 : (1 + 1/15 + 1/15 + 1/15 + 2/15 + + 4/15 + 6/15) at temperatures of 25, 15 or 5 degrees C. The dissolved ejaculate was equilibrated for 15 minutes, three minutes it was left to freeze in liquid nitrogen vapours and 24 hours it was stored at a temperature of -196 degrees C. Motility, percentage of live spermatozoa and percentage of morphologically normal spermatozoa were evaluated after sample thawing at 15 degrees C. The quality of cryo-preserved spermatozoa of stags was most effectively influenced by the addition of ethylene glycol to ejaculate at the concentration of 0 + 16% by the method of 1 : (1 + 1/15 + 1/15 + + 1/15 + 2/15 + 4/15 + 6/15) at the temperature of 25 degrees C.     

The effect of storage conditions on samples for the evaluation of copper status in blesbok (Damaliscus pygargus phillipsi)

Investigaltions to determine the effect of sample storage on the concentration of copper in liver tissue and on the activity of erythrocyte superoxide dismutase were undertaken in preparation for a study of blesbok (Damaliscus pygargus phillipsi) that were suspected to be suffering from copper deficiency. Two liver samples were collected from each of 20 culled blesbok in a manner that simulated the collection of biopsies from the live animal. These samples were stored either in 10% formalin or frozen at -20 degrees C until analysed 4 1/2 months later. The effect of different methods of sample storage on superoxide dismutase activity was determined. Erythrocytes collected from 3 Jersey cows and 5 culled blesbok were washed and divided into 0.5 ml portions, stored at room temperature (approximately 20 degrees C), in a refrigerator (4 degrees C), frozen at -20 degrees C in a freezer, and in liquid nitrogen (-200 degrees C). An analysis of superoxide dismutase activity was undertaken using a commercial assay kit at intervals of 2-4 days until the levels of activity had fallen significantly. The copper concentration in formalin-preserved liver samples was significantly lower than that measured in frozen liver tissue apparently as a result of leaching. The activity of superoxide dismutase in cattle blood was unchanged for 4 days at room temperature but fell appreciably after 2 days at 4 degrees C and -20 degrees C. Enzyme activity remained unchanged for 200 days in erythrocytes stored in liquid nitrogen. Superoxide dismutase activity levels in healthy blesbok were considerably lower than those measured in Jersey cows and remained unaffected for up to 6 days in samples stored at 4 degrees C and 20 degrees C. The level of activity fell significantly thereafter. Samples stored in liquid nitrogen were unchanged after 40 days.     

A high efficiency technique for the long-term preservation of infective nematode larvae.

Improvements are suggested for the existing long term techniques for the preservation of nematode larvae. Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis and Cooperia curticei larvae exsheathed in sodium hypochlorite and then suspended in phosphate buffered saline (PBS pH 7.2) are cooled in the gas over liquid nitrogen at a cooling rate of -1 degree C min-1 down to -50 degrees C. Larvae are then stored in liquid nitrogen at -196 degrees C. After warming at 30 degrees C and reactivation at 20 degrees C for at least 12 h, their percent motility is maintained (approximately 85%) providing that no more than 3000 to 5000 larvae are suspended in 1.8 mL of PBS in cryotubes. Infectivity does not significantly decrease: 46% of larvae cooled for 2 or 6 mo develop to adult stages compared to 52% for larvae stored at 4 degrees C for 2 mo.     

Effect of temperature on the growth of Penicillium from moldy feed

178 Penicillium strains were isolated from moulded feedstuffs (mainly corn and wheat after ambient air drying, wheat after refrigeration, corn after treatment with propionic acid), and investigated for the effect of temperature on growth rate. Temperatures between 4 and 27 degrees C were used. 159 strains proved psychrotrophic, they were able to grow at 4 degrees C on malt extract agar (MA) as well as in wheat; the growth in wheat was indicated by the production of ergosterol. On MA all psychrotrophic strains were able to grow also at 10-27 degrees C. The maximum rate of growth was reached at 10-15 degrees C, 15-20 degrees C, 20-27 degrees C, or must be assumed for temperatures greater than or equal to 27 degrees C. At 4 and 10 degrees C the mean growth rate on MA was 24 and 51%, respectively, related to the mean rate at 20 degrees C; the lower temperature limit of growth was found at - 1 degree C by graphic extrapolation. The Q10 value of growth on MA is about 2, if temperatures between 20 and 10 degrees C are compared; with decreasing temperature higher Q10 values up to 3.7 are obtained. The growth rate of a strain of Penicillium aurantiogriseum on wheat was affected by temperature nearly in the same manner as on malt extract agar. The results are discussed with respect to the risk of moulding during refrigerated storage of feedstuffs.     

投入液氮前的平衡温度及时间对兔胚冻后存活的影响

本研究旨在了解投入液氮前的平衡温度及时间对兔胚冻后存活的影响。经0.5M蔗糖与1.5,2.0或2.5M乙二醇组成的冷冻液处理后的兔胚,在-30℃或-37℃平衡10或20分钟后即投入液氮保存或直接投入液氮保存。胚胎解冻在37℃的水浴进行。当平衡温度由-30℃下降到-37℃时,胚胎的冻后形态正常率及存活率均极显著下降(P<0.01)。在-30℃平衡20分钟与10分钟的冻后存活率无明显差异(P>0.05),但直接投入液氮时,冻后存活率则很低(8.3％)。由此表明:兔胚冻前在-30℃平衡10—20分钟是必要的。     

The effect of temperature and relative humidity on survival of eggs of the cestode, Monieza expansa (Rudolphi, 1810)]

The survival of Moniezia expansa eggs in the droppings of lambs was investigated at various temperatures in laboratory conditions and on test plots outdoors. The optimum temperature of the livability of M. expansa eggs in laboratory conditions is 5 degrees C; at this temperature 10% of oncospheres survived after 161 days. At the temperatures of 10, 25, 35 degrees C the oncospheres survived 105, 28, 46 days respectively, at -12 degrees C it was 28 days. It was for 21 and 35 days that on the test grassy plots the eggs of M. expansa survived in the droppings of lambs in the summer months of July and August at the average air temperatures of 15.7-18.2 degrees C and relative humidity of 67.7-74.3%. In autumn in September and October, at the average temperatures of 5.8-14.6 degrees C and relative humidity of 65.3-76.7% the oncospheres survived for 49 to 91 days. The M. expansa eggs in the droppings of lambs were able to survive on the test plot. The living oncospheres were demonstrated for 119 days from November 1987 to March 1988, and for 175 days till May by means of experimental infection of intermediate hosts.     

Freezing of sheep faeces invalidates Haemonchus contortus faecal egg counts by the McMaster technique

Faecal pellets from a sheep that was artificially infected with a monoculture of Haemonchus contortus were collected over a 2-h period in the morning. In the laboratory the faeces were thoroughly mixed by hand and 48 by 1 g aliquots of the pellets were sealed in plastic bags, from which the air had gently been expressed. The faecal worm egg count of the sheep was about 14,000 g(-1). Varying numbers of the bags were either processed for faecal worm egg counting (FEC) by the McMaster technique on day 0, or were stored at one of the following temperatures: about 4 degrees C, -10 degrees C or -170 degrees C before processing. The faecal aliquots that were frozen were thawed at room temperature after having been frozen for either 2 h or 7 days, and processing of aliquots maintained at 4 degrees C proceeded shortly after the samples had been removed from the refrigerator. A dramatic reduction in egg numbers was found in all the aliquots that were frozen at -170 degrees C before faecal worm egg counts were done, as well as in those frozen for 7 days at about -10 degrees C. Numerous empty, or partially empty, egg shells were observed when performing the counts in faeces that had been frozen. In contrast, there was no significant reduction in the numbers of eggs in aliquots maintained for 7 days in a refrigerator at +/- 4 degrees C before examination, when compared with others examined shortly after collection of the faeces. Since H. contortus eggs in faeces are damaged by freezing, some methods that can be used for short term preservation are outlined. It is concluded that all nematode egg counts from cryopreserved faeces (whether in a freezer at -10 degrees C or in liquid nitrogen) should possibly be regarded as being inaccurate, unless the contrary can be demonstrated for different worm genera. However, exceptions are expected for the more rugged ova, such as those of the ascarids and Trichuris spp.     

Effects of syringe material and temperature and duration of storage on the stability of equine arterial blood gas variables

OBJECTIVES: To evaluate the consistency of partial pressures (P) of arterial oxygen (aO(2)), arterial carbon dioxide (aCO(2)) and pH measurements in equine carotid arterial blood samples taken into syringes made from three different materials and stored at room temperature or placed in iced water for measurement at three different times. STUDY DESIGN: Prospective observational study over 19 days. ANIMALS: Four clinically normal Thoroughbred or Thoroughbred-cross horses (three geldings, one mare, mean age 6.25 years, range 5-7 years). METHODS: Identical blood samples were taken on two separate occasions from the carotid arteries of the four horses into syringes made of glass, plastic and polypropylene. PaO(2), PaCO(2) and pH determinations were performed on blood from each syringe type at 10, 60 and 120 minutes post-sampling with samples stored at room temperature (approximately 20 degrees C) or in iced water (approximately 0 degrees C). Data were analysed by anova and a split plot model fitting syringe within horse X pair and time within temperature within syringe. RESULTS: Syringe material, storage temperature and time before analysis all had significant effects on PaO(2) (p < 0.001). PaCO(2) was unaffected by syringe material or storage temperature. However, over 120 minutes, storage duration significantly (p = 0.002) affected values. Temperature of storage and duration prior to analysis both significantly affected pH values (p = 0.005 and p < 0.001, respectively), but syringe material did not. Several significant interactions between these variables were noted. CONCLUSIONS: Equine arterial blood gas determination has a different sensitivity to storage conditions compared to other veterinary species. CLINICAL RELEVANCE: For accurate equine arterial blood analysis, PaO(2) samples need to be analysed within 10 minutes or taken into glass syringes, stored on ice and analysed at 2 hours post-sampling. PaCO(2) and pH measurements can be performed on samples stored in glass, plastic or polypropylene syringes at room temperature for up to 1 hour post-sampling.     

Successful use of deep-frozen stallion sperm after 23 years of storage at -196 degrees C

A reduction in the motility of the spermatozoa in stallion semen stored in pellet form for 23 years in liquid nitrogen at -196 degrees C could not be seen after thawing. The insemination of a mare with this semen resulted in a normal pregnancy. A normally developed, healthy male foal was born after a gestational period of 321 days.     

Changes in gas composition and acid-base values of venous blood samples stored under different conditions in 4 domestic species

BACKGROUND: The effect of storage temperature and time on blood gas and acid-base values has been investigated intensively in cattle and dogs; however, data are lacking in other species. OBJECTIVE: The aim of our study was to evaluate changes in gas composition and acid-base values in venous blood stored at different temperatures and for different times in 4 domestic species in Italy. METHODS: Blood samples from Comisana sheep (n = 10), Maltese goats (n = 10), Ragusana donkeys (n = 10), and Thoroughbred horses (n = 10) were analyzed after storage at 23 degrees C (room temperature) for 15 minutes (group I), 23 degrees C for 1 hour (group II), 37 degrees C for 8 hours (group III), and 4 degrees C for 24 hours (group IV). Results were analyzed using a 1-way repeated measures ANOVA. RESULTS: In all species no statistically significant differences in pH values were present in samples stored at 4 degrees C for 24 hours. This also was true for PCO2 in all species except the horse. Except for HCO3- concentration in the horse, significant changes in PO2, HCO3- concentration, base excess, and the standard bicarbonate concentration were observed for all species in samples stored at 4 degrees C. In samples stored for only 1 hour at room temperature, significant changes in most analytes were detected. CONCLUSIONS: The results of this study underline the need for rapid assessment of acid-base samples, because any delay, even for 1 hour, may affect the results.     

Activity of supplemental enzymes and their effect on nutrient utilization and growth performance of growing chickens as affected by pelleting temperature.

Activity of supplemental enzymes in a barley-soybean-maize based diet at 60, 75 and 90 degrees C pelleting temperatures was studied using feed viscosity, in-vitro enzyme activity and broiler performance data. High pelleting temperatures increased feed viscosity but supplemented enzymes reduced the viscosity at all three temperatures levels by 11, 14 and 17%, respectively. Water intake and losses in excreta of birds were found to be affected by feed viscosity. Activity of cellulase enzyme, measured using the radial diffusion method, was unaffected at 60 and 75 degrees C, but reduced by 73% in feed processed at 90 degrees C. Enzymes increased the weight gain of broilers by 11.1% at 90 degrees C, but no effect could be seen at low pelleting temperatures possibly due to high dietary protein and energy contents. Feed intake was unaffected by enzymes. Birds consumed 6% more feed and grew 9% faster when the pelleting temperature was increased from 60 to 75 degrees C. Reduced feed intake and daily weight gain observed at 90 degrees C could be fully compensated by the enzyme supplementation. High pelleting temperature reduced energy metabolizability (3.2%) and nitrogen utilization (4%) but enzyme almost compensated them (by 3.3% and 2.6%, respectively). No interaction could be detected between the pelleting temperatures and enzymes. It is concluded that pelleting temperatures as high as 90 degrees C drastically reduce cellulase activity, energy and nitrogen utilization thus lowering broiler performance. Either the remaining activity of cellulase or other thermostable enzymes can prevent the losses.     

Aerosol stability of bovine parainfluenza type 3 virus.

Aerosols of bovine parainfluenza type 3 virus were generated with a Devilbiss 40 nebulizer from Eagle's minimum essential medium and nasal secretion from a non-infected calf and stored in a rotating drum at temperatures of 6 degrees C or 32 degrees C and relative humidities of 30% or 90%. The aerosols were sampled at seven minutes, one, two and three hours after the start of generation with an all glass impinger (AGI-30) and titrated for infectivity in cell cultures. Physical decay was determined by a rhodamine tracer technique. Media, temperature or relative humidity had little effect on the survival of parainfluenza type 3 virus during spraying (zero to seven minutes). During aging of aerosols at 32 degrees C and 30% relative humidity, parainfluenza type 3 virus was less stable in Eagle's minimum essential medium than in nasal secretion from a noninfected calf, but at 6 degrees C and 30% relative humidity, the virus was more stable in Eagle's minimum essential medium. At 32 degrees C, the virus was less stable during aging at 90% relative humidity than at 30% relative humidity. The virus was consistently more stable during aging of aerosols at 6 degrees C than at 32 degrees C.     

A thermal threshold testing device for evaluation of analgesics in cats

A thermal analgesiometric device was developed for unrestrained cats. Heat was provided by an electrical element potted together with a temperature sensor in thermally conductive epoxy in a 5 gm probe. This was attached to an elasticated band round the cat's thorax with an inflated bladder maintaining constant pressure between probe and skin. A safety cut-off was set at 60 degrees C. End point was a skin flick, turning, or jumping. Threshold temperatures in untreated cats were around 40 degrees C and repeatable to 4 degrees C with 5, 10 or 15 minutes between tests. Threshold temperature was stable in tests at 15 minutes intervals without false positives or negatives. Tests repeated at weekly intervals were repeatable to within 4 degrees C. Treatment with the opioid analgesic pethidine increased the threshold temperatures 10.2 (6.7) degrees C 45 minutes after treatment. The device was well tolerated for at least 24 hours and the analgesic effect of an opioid was detected. The system appears suitable for use in investigations into analgesic pharmacology in cats.     

Preservation of fowl semen in liquid nitrogen--an improved method

1. An improved method of storing fowl semen in liquid nitrogen is described. It uses glycerol as a cryopotectant and the degree of fertility obtained with semen stored for up to 2 1/4 years was very promising. 2. The essential features of the method are a slow freezing rate, a reduction of temperature to - 35 degrees C before the semen is plunged into liquid nitrogen and insemination within 15 min of thawing and removing the glycerol from the diluent. 3. It seems likely that further improvements in the technique may be expected through better control of the injection of liquid nitrogen into the freezing chamber, thereby minimising localised, rapid temperature changes around the ampoules during freezing. 4. Preliminary results suggest that it may prove possible to select males for the ability of their spermatozoa to withstand freezing. 5. The importance of recognising differences between males in the initial quality of semen and between females in the physiology of the reproductive organs in determining the fertilising ability of stored semen is discussed.     

Effects of storage time and temperature on pH,specific gravity,and crystal formation in urine samples from dogs and cats

OBJECTIVE: To determine effects of storage temperature and time on pH and specific gravity of and number and size of crystals in urine samples from dogs and cats. DESIGN: Randomized complete block design. ANIMALS: 31 dogs and 8 cats. PROCEDURE: Aliquots of each urine sample were analyzed within 60 minutes of collection or after storage at room or refrigeration temperatures (20 vs 6 degrees C [68 vs 43 degrees F]) for 6 or 24 hours. RESULTS: Crystals formed in samples from 11 of 39 (28%) animals. Calcium oxalate (CaOx) crystals formed in vitro in samples from 1 cat and 8 dogs. Magnesium ammonium phosphate (MAP) crystals formed in vitro in samples from 2 dogs. Compared with aliquots stored at room temperature, refrigeration increased the number and size of crystals that formed in vitro; however, the increase in number and size of MAP crystals in stored urine samples was not significant. Increased storage time and decreased storage temperature were associated with a significant increase in number of CaOx crystals formed. Greater numbers of crystals formed in urine aliquots stored for 24 hours than in aliquots stored for 6 hours. Storage time and temperature did not have a significant effect on pH or specific gravity. CONCLUSIONS AND CLINICAL RELEVANCE: Urine samples should be analyzed within 60 minutes of collection to minimize temperature- and time-dependent effects on in vitro crystal formation. Presence of crystals observed in stored samples should be validated by reevaluation of fresh urine.     

Influence of the Freezing Technique (Nitrogen Liquid vs Ultrafreezer of −152°C) and Male-to-Male Variation Over the Semen Quality in Canarian Mastiff Breed Dogs

This study was carried out to assess the in vitro quality of canine semen frozen in an ultrafreezer at -152 degrees C and to evaluate the male-to-male variation of frozen semen in five male dogs of the Canarian Mastiff breed. Four ejaculates of each dog were processed individually (5% glycerol and 0.5% Equex) to reach a final concentration of 100 x 10(6) spermatozoa/ml. Then, two freezing techniques were tested to assess the seminal quality (sperm motility, live spermatozoa and abnormal sperm cell percentages) at 1, 30, 60, 120 and 360 days after freezing: (i) semen was frozen and stored in liquid nitrogen; (ii) semen was frozen and stored in the ultrafreezer at -152 degrees C. After freezing-thawing, both freezing protocols showed no significant differences in sperm motility and the percentages of live and abnormal spermatozoa. On the other hand, the microscopic characteristics of spermatozoa in fresh semen were practically similar among males; however, after the semen processing and freezing, significant differences were observed (p < 0.05) among males, especially as regards sperm motility. This inter-individual variability was detected in both freezing protocols, showing that the male-to-male variation in the seminal quality post-freezing was independent of the freezing technique used. The in vitro results obtained in the Canarian Mastiff breed confirmed that the use of ultra-freezers at -152 degrees C is a potential alternative to liquid nitrogen for storing canine semen for long periods of time.     

Stability of glutathione peroxidase in swine plasma samples under various storage conditions.

The stability of plasma glutathione peroxidase under different temperatures (4 degrees C vs. -15 degrees C), various durations of storage (0, 1, 2, 3, 7, 14, 28 and 56 d), and storage under inert gas (nitrogen (N2)) vs air is described. The glutathione peroxidase activity of swine plasma decreased consistently with storage at either 4 degrees C or -15 degrees C 1-56 d after collection, and differed (P less than or equal to 0.01) from the initial values. Storage under N2 at -15 degrees C slowed the rate of enzyme activity decrease but did not maintain the initial activity. For absolute measurements, it is suggested that swine plasma glutathione peroxidase activity be measured immediately after separation from the blood cells or be assayed within 24 h in plasma samples stored at -15 degrees C with air space displaced by N2. If relative treatment differences in enzyme activity are satisfactory, then assays can be conducted after controlled periods of storage.     

Effects of storage temperature and time on canine plasma ammonia concentrations

Stability of ammonia in canine plasma was determined, with regard to temperature and time of storage. Heparinized venous blood samples were collected from 8 healthy dogs, immediately placed in an ice water bath, and centrifuged at 5 C. Plasma was harvested from the blood samples, and the initial analysis of each sample for plasma ammonia was performed within 30 minutes after collection. Separate aliquots of the plasma from each dog were stored at 21 C, 4 C, -15 C, or -40 C. Ammonia concentrations of the aliquots stored at the various temperatures were determined at 24, 48, and 96 hours after collection. Statistical analysis of the data from each dog did not indicate a significant relationship between the initial concentration of plasma ammonia and subsequent determinations. Correlation or significance was not found among samples stored at similar temperatures and evaluated at similar times.