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.     

Stability and storage characteristics of enzymes in cattle blood

The stability and storage characteristics were studied of 11 bovine enzymes of potential clinical significance, namely, aldolase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, acetylcholinesterase, creatine kinase, gamma glutamyltransferase, glutathione peroxidase (GSH-Px), alpha-hydroxybutyrate dehydrogenase, lactate dehydrogenase and superoxide dismutase (SOD). Enzyme activities in fresh serum were compared with those in plasma containing various anticoagulants including lithium heparin, EDTA and oxalate/fluoride. The same preservatives were assessed for their effects on the whole blood activities of GSH-Px and SOD. Stabilities of enzymes in plasma and serum stored at room (+20 degrees C), refrigerator (4 degrees C) or deep freeze (-20 degrees C) temperatures were also compared. In addition, SOD and GSH-Px activities in samples stored, at the same temperatures, as whole blood or aqueous lysates were monitored.     

Effects of low and high temperatures on infectivity of Cryptosporidium muris oocysts suspended in water

Cryptosporidium muris oocysts suspended in 200 microl of water were pipetted into plastic microcentrifuge tubes which were stored at 4 degrees C or frozen at -5 degrees C for 1, 3, 5, 7, and 10 days and at -20 degrees C for 1, 3, 5, and 8h, respectively. Other samples of C. muris oocysts suspended in water were heated in the metal block of a thermal DNA cycler. Block temperatures were set at 5 degrees C incremental temperatures from 40 to 70 degrees C. At each high temperature setting microcentrifuge tubes containing C. muris oocysts were exposed for 1 min. Both, frozen and heated oocyst suspensions as well as untreated control oocyst suspensions were then inoculated into each of four ICR mice by gastric intubation. Untreated, freeze-thawed or heated oocysts were considered infectious when oocysts of C. muris were found microscopically in the faeces of mice after inoculation. All inoculated mice that received oocysts frozen at -5 degrees C for 3, 5, 7, and 10 days and -20 degrees C for 1, 3, 5, and 8h had no oocysts in faeces. In contrast, C. muris oocysts frozen at -5 degrees C for 1 day remained infective for inoculated mice. Our results also indicated that when water containing C. muris oocysts was exposed at a temperature of 55 degrees C or higher for 1 min, the infectivity of oocysts was lost.     

The effect of temperature on growth and survival of Fusobacterium necrophorum isolated from bovine liver abscesses.

The ability of Fusobacterium necrophorum to survive or grow in liquid nitrogen or at temperatures between -10 degrees and 59 degrees C was determined. The organism remained viable but did not grow in liquid nitrogen or between -10 degrees and 21 degrees C. It grew between 22 degrees and 43 degrees C. No isolate grew at temperatures above 43 degrees C and all three isolates survived for a minimum of 15 minutes and an average of 25 minutes at 59 degrees C. The optimum temperature for maximum growth was 37 degrees C. The organism survived in ampoules stored in liquid nitrogen for eight years. It survived in liver abscesses stored at -10 degrees C for five years and as cultures in screw capped tubes for three years.     

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 and storage characteristics of enzymes in sheep blood

The stability and storage characteristics were studied of 11 ovine enzymes of potential clinical significance, namely, aldolase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, acetylcholinesterase, creatine kinase, gamma glutamyltransferase, glutathione peroxidase (GSH-Px), alpha-hydroxybutyrate dehydrogenase, lactate dehydrogenase and superoxide dismutase (SOD). Enzyme activities in fresh serum were compared with those in plasma containing various anticoagulants including lithium heparin, EDTA and oxalate/fluoride. The same preservatives were assessed for their effects on the whole blood activities of GSH-Px and SOD. Stabilities of enzymes in plasma and serum stored at room (+20 degrees C), refrigerator (4 degrees C) or deep freeze (-20 degrees C) temperatures were also compared. In addition, SOD and GSH-Px activities in samples stored, at the same temperatures, as whole blood or aqueous lysates were monitored. The results are discussed with particular reference to the differences between sheep and cattle.     

Quantification of Ca2(+)-dependent protease activities by hydrophobic and ion-exchange chromatography

Hydrophobic and ion-exchange chromatography were compared for yield of Ca2(+)-dependent proteases and their inhibitor in studies designed to quantify Ca2(+)-dependent proteases activity for comparative purposes. Ion-exchange (DEAE-Sephacel) proved superior to hydrophobic chromatography (Phenyl-Sepharose). Under the proper conditions, DEAE-Sephacel effectively separated low-calcium-requiring form of Ca2(+)-dependent protease (CDP-I) and CDP inhibitor. Characterization of the assay system for components of the Ca2(+)-dependent proteolytic system separated by ion-exchange chromatography indicated that proteolytic degradation of casein by Ca2(+)-dependent proteases was linear with time for up to 60 min at 25 degrees C and that it was linear up to .4 to .45 units of activity. Therefore, we recommend that, after identification of fractions containing Ca2(+)-dependent protease (CDP-I or CDP-II), these fractions be pooled, and reassayed at a volume that yields values of less than .45 units of activity. Unlike CDP-I and CDP-II, CDP inhibitor lost its activity rapidly with frozen storage (frozen in liquid nitrogen, then stored at -70 degrees C); therefore, inhibitor should be assayed in fresh (unfrozen) samples only.     

Viability and infectivity of Trichinella spiralis muscle larvae in frozen horse tissue

Many aspects of the biology and epidemiology of Trichinella infection in the horse are poorly understood, including survival of Trichinella spp in horse muscle. In this study, we have assessed the freeze tolerance of T. spiralis in horse meat stored at 5, -5, and -18 degrees C for 1 day to 24 weeks. Results demonstrate a steady reduction in the number of live ML recovered from the cold stored meat samples. On Day 1, recovery of live larvae had been reduced by 18.6%, 50.1%, and 37.2%, and by 4 weeks, recovery of larvae had been reduced by 65.4%, 66.5%, and 96.2% in samples stored at 5, -5, and -18 degrees C, respectively. Infectivity results (measured as reproductive capacity index (RCI)) from mice inoculated with larvae recovered from non-frozen meat samples at day 0 was 23.5. Following storage at -18 degrees C for one and two days, the RCIs were 2.09 and 0.99, respectively. Small numbers of infective larvae were still present in meat samples stored at -18 degrees C for 4 weeks. The RCI of ML recovered from meat samples stored at -5 degrees C was 14.99 and 6.36 at 2 weeks and 4 weeks respectively; the RCI of samples stored at 5 degrees C was 23.1 at 8 weeks, and fell rapidly thereafter (12 week RCI 1.33; 0 at 24 weeks). These data demonstrate that infective T. spiralis, a non-freeze tolerant species, can survive for at least 4 weeks in horse tissue frozen at -5 or -18 degrees C, and that the numbers of infective larvae decrease substantially by day 2 at -18 degrees C and by week 4 at -5 degrees C.     

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.     

Infectivity of cryopreserved Babesia bovis, Babesia bigemina and Anaplasma centrale for cattle after thawing, dilution and incubation at 30 degrees C

Blood containing either Babesia bovis, Babesia bigemina or Anaplasma centrale was mixed with an equal volume of 3 M glycerol in phosphate-buffered saline with or without glucose and then stored in liquid nitrogen for 2-30 days. After being thawed, the parasitized blood was subjected to various procedures, including dilution up to 1000-fold followed by incubation at 30 or 4 degrees C for 8 h, before infectivity of the parasites was tested in a total of 70 cattle. The results showed that the blood cryopreserved with glycerol remained highly infective after thawing, despite dilution and incubation for 8 h at 30 degrees C. The results have practical application in the use of frozen, live vaccines against bovine babesiosis and anaplasmosis.     

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.     

Effect of storage of reconstituted recombinant human thyroid-stimulating hormone (rhTSH) on thyroid-stimulating hormone (TSH) response testing in euthyroid dogs

Bovine thyrotropin (bTSH) stimulation testing has long been considered the gold standard for diagnosis of canine hypothyroidism. Unfortunately, bTSH is no longer commercially available. Recently, the use of recombinant human thyrotropin (rhTSH) to perform thyroid-stimulating hormone (TSH) stimulation testing in dogs was described. The cost of an rhTSH vial (1.1 mg) limits the practical use of this product. The study reported here was performed to determine the effects of storing rhTSH on the post-TSH increase of serum total (TT4) and free (FT4) thyroxine concentrations during TSH stimulation testing in 12 euthyroid Beagles in a crossover trial. Three TSH tests with recombinant human thyrotropin (rhTSH; 91.5 microg IV) were performed on each dog during 3 different periods: 1 with freshly reconstituted rhTSH (fresh); 1 with rhTSH, reconstituted and stored at 4 degrees C for 4 weeks (refrigerated); and 1 with rhTSH, reconstituted and frozen at -20 degrees C for 8 weeks (frozen). Blood samples for determination of TT4 and FT4 concentrations were collected before and 4 and 6 hours after rhTSH administration. There was no significant difference in TT4 or FT4 concentration after stimulation with fresh, refrigerated, and frozen rhTSH. Furthermore, there was no significant difference between TT4 or FT4 serum concentration observed 4 and 6 hours after rhTSH administration. In conclusion, reconstituted rhTSH can be stored at 4 degrees C for 4 weeks and at -20 degrees C for 8 weeks without loss of biological activity, allowing clinicians to perform more TSH response tests per vial.     

聚乙烯吡咯烷酮对公猪精液冷冻的影响

试验旨在评价聚乙烯吡咯烷酮（polyvinylpyrrolidone,PVP）对公猪精液冷冻的影响。试验分为5组,分别为对照组（不添加PVP）和PVP处理组（在冷冻基础液中分别加入0.25%、0.50%、1.00%、2.00% PVP）。采用手握法采集松辽黑猪精液,用5种冷冻基础液稀释,在25 ℃平衡1 h,17 ℃平衡2 h,4 ℃平衡3 h后灌装于0.5 mL细管中,在液氮上方3 cm处熏蒸10 min,保存在液氮罐中30 d后进行检测。样本解冻后分别检测精子活力、质膜完整性、顶体完整性、线粒体活性、DNA完整性、过氧化氢酶（catalase,CAT）活性、超氧化物歧化酶（superoxide dismutase,SOD）活性、谷胱甘肽过氧化物酶（glutathioneperoxidase,GSH-Px）活性、活性氧簇（reactive oxygen species,ROS）水平及丙二醛（malondialdehyde,MDA）水平。结果显示,与对照组相比,冷冻基础液中添加0.50% PVP显著提高冻融后精子的活力、质膜完整性、顶体完整性、线粒体活性、CAT活性、SOD活性、GSH-Px活性（P<0.05）,显著降低精子ROS和MDA水平（P<0.05）;与对照组相比,添加PVP有利于提高DNA完整性,但差异不显著（P>0.05）。因此,猪精液冷冻基础液中添加PVP可改善冻融后精子质量,添加0.50%效果最佳。     

Stability of canine factor VIII: coagulant activity in vitro.

A pilot study was undertaken to assess the stability of canine factor VIII:coagulant (FVIII:C) activity over three days, under various storage conditions (plasma at 4, 20 and 37 degrees C, whole blood at 4 and 20 degrees C). Blood collected from normal and hemophiliac dogs was used. Both plasma and whole blood samples appeared to be stable for up to 48 h at 4 and 20 degrees C. A subsequent study evaluated FVIII:C stability at 4 and 20 degrees C when stored as whole blood only. Samples were tested at 0, 24 and 48 h after collection. At 4 degree C there was a significant decline at 24 h (p less than 0.05), from 110% to 97% (mean values). Although the mean value was further decreased at 48 h (89%) this was not significant (p greater than 0.05). No significant change in FVIII:C activity was observed in whole blood stored at 20 degrees C for 24 or 48 h (110% and 107% respectively). These results suggest that canine whole blood samples collected into sodium citrate stored at 20 degrees C are adequate for routine FVIII:C assay for up to 48 h after collection.     

Effect of freezing goat milk samples on recovery of intramammary bacterial pathogens

With the aim of evaluating the effect of freezing goat milk samples on recovery of intramammary pathogens, 1200 milk samples from udder halves with subclinical intramammary infection were studied. Samples (20 ml) were frozen at -20 and at -80 degrees C. Thawing was carried out at room temperature at 7, 14, 21, 28, 58, 118, 178, 236 and 730 days after collection and bacteriological analyses were carried out to determine the number of colony forming units/ml (CFU/ml). Mixed model statistical analysis showed that bacterial group, temperature of storage, interaction of bacterial group and temperature of storage and the interaction of bacterial group, time and temperature of storage were statistically significant effects. For coagulase negative staphylococci (CNS), least squares means of log CFU/ml recovered at -20 and -80 degrees C were not different. Nevertheless, for Gram negative bacilli (GNB) a significant decrease was detected in samples frozen at -20 vs. -80 degrees C. At both temperatures and at different times of storage, significant increases were detected between log CFU/ml of CNS and values on day zero. At -20 degrees C, a significant decrease in GNB recovery was detected between freezing days zero and 730. This difference was not detected when goat milk samples infected by GNB were frozen at -80 degrees C. The results show that frozen milk samples can be useful in goat subclinical mastitis control programs.     

Effects of storage conditions in vitro on the assay of chick liver glycogen.

1. The reproducibility of an enzymic method for assaying chick liver glycogen was found to be +/- 1.6%. 2. Storage of liver at either -21 or -70 degrees C for up to 4 weeks, after freezing in liquid nitrogen, had no significant effect on glycogen concentration. However, the concentration of free glucose increased by about 50% during this time. 3. Storage of liver in ice-cold saline for up to 3 h proved unsatisfactory because of a significant loss of glycogen. 4. There was no significant difference in immediate glycogen concentration in liver tissue that was freeze-clamped, frozen in liquid nitrogen or collected into ice-cold saline. The free glucose concentration was higher, when estimated immediately after, with the latter two methods of collection of liver tissue.     

Ion-exchange microchromatography and thiobarbituric acid colorimetry for the measurement of canine glycated hemoglobins

Nonenzymatic glycation of hemoglobin is a slow, continuous, and irreversible process which takes place during the whole lifespan of the erythrocyte. When hemolytic diseases are ruled out, the levels of glycated hemoglobins reflect the time-averaged serum glucose concentration for the preceding weeks. Canine hemoglobin also binds physiologically to intraerythrocytic glucose to form a glycated fraction which provides information on the animal's long-term glycemic status. This study describes an overall evaluation of ion-exchange microchromatography and thiobarbituric acid (TBA) colorimetry for the measurement of canine glycated hemoglobins. The intra- and inter-assay coefficients of variation (CVs) found were less than 5% in normal and diabetic canine samples, and both assays proved linear over the analytical range tested, which was wide enough to include the expected clinical values. Under our laboratory's conditions, the reference range for HbA(1) was 5.82 +/- 0.62% and for HbA(1)c was 2.35 -/+ 0.47%. Sample stability was lower using the ion-exchange procedure, with increases in HbA(1) observed after 4 days in whole blood and hemolysates stored at room temperature, after 12 days in whole blood stored at 4 degrees C, and after 7 days in hemolysates stored at 4 degrees C and -20 degrees C. In the case of TBA colorimetry, whole blood was stable for at least 21 days at room temperature and at 4 degrees C, and hemolysates were stable for 18 days at room temperature, at least 21 days at 4 degrees C, and up to 3 months at -20 degrees C.     

The persistence of bovine enterovirus and pseudorabies virus in liquid cattle manure at different storage temperatures

The tenacity of viruses in liquid manure of cattle was examined in a total of five samples inoculated with ECBO-virus (strain LCR-4) representing viruses without envelope and Aujeszky virus (field isolate) representing enveloped viruses. The titers were examined at regular intervals over a period of 26 weeks. On the day of inoculation each sample had a titer of 10(5) ID50/ml. After 16 weeks complete inactivation was observed in the Aujeszky virus sample stored at 20 degrees C. The Aujeszky virus sample wich was kept at 4 degrees C at 26 weeks had a titer of 10(1,75) ID50/ml. In the samples inoculated with ECBO virus after 26 weeks of inoculation a titer of 10(3) ID50/ml was found in the manure stored at 20 degrees C. No influence on the virus titers in the liquid manure samples was observed either from pH or the number of bacteria (3,4 x 10(7)-1.16 x 10(8)/ml during the examination period.     

Effects of season and sample handling on measurement of plasma alpha-melanocyte-stimulating hormone concentrations in horses and ponies

OBJECTIVE: To investigate effects of sample handling, storage, and collection time and season on plasma alpha-melanocyte-stimulating hormone (alpha-MSH) concentration in healthy equids. ANIMALS: 11 healthy Standardbreds and 13 healthy semiferal ponies. PROCEDURE: Plasma alpha-MSH concentration was measured by use of radioimmunoassay. Effects of delayed processing were accessed by comparing alpha-MSH concentrations in plasma immediately separated with that of plasma obtained from blood samples that were stored at 4 degrees C for 8 or 48 hours before plasma was separated. Effects of suboptimal handling were accessed by comparing alpha-MSH concentrations in plasma immediately stored at -80 degrees C with plasma that was stored at 25 degrees C for 24 hours, 4 degrees C for 48 hours or 7 days, and -20 degrees C for 30 days prior to freezing at -80 degrees C. Plasma alpha-MSH concentrations were compared among blood samples collected at 8:00 AM, 12 noon, and 4:00 PM. Plasma alpha-MSH concentrations were compared among blood samples collected in January, March, April, June, September, and November from horses and in September and May from ponies. RESULTS: Storage of blood samples at 4 degrees C for 48 hours before plasma was separated and storage of plasma samples at 4 degrees C for 7 days prior to freezing at -80 degrees C resulted in significant decreases in plasma alpha-MSH concentrations. A significantly greater plasma alpha-MSH concentration was found in September in ponies (11-fold) and horses (2-fold), compared with plasma alpha-MSH concentrations in spring. CONCLUSIONS AND CLINICAL RELEVANCE: Handling and storage conditions minimally affected plasma alpha-MSH concentrations. Seasonal variation in plasma alpha-MSH concentrations must be considered when evaluating pituitary pars intermedia dysfunction in equids.     

Determination of the number of somatic cells in milk using the rapid diphenylamine DNA filter method

The rapid reaction of the diphenylamine agent with DNA was used for the determination of the counts of somatic cells in cow's milk, using the DNA filter method. The method is based on the filtration of a warmed (65-70 degrees C) mixture of milk with Triton X-100 through the Synpor nitrocellulose membrane filter, pore size 2 to 5 microns, and subsequent DNA determination of the collected somatic cells by the colour reaction of diphenylamine. A 2ml quantity of distilled water and 4 ml of diphenylamine reagent were added to the membrane filters with somatic cells. The mixture is warmed in water bath at 90 to 100 degrees C for 20 min., then it is cooled, centrifuged (3500 X g, 15 min.), and the optical density is measured at 595 nm. The relation 8 micrograms = 1 million cells was used for the conversion of DNA content to the counts of cells. The average variation coefficient of the determination was 5.9% and the coefficient of correlation between the diphenylamine DNA filter method and the direct microscopy of the somatic cells on membrane filters was r = 0.997. Using the diphenylamine DNA filter method, the counts of somatic cells can also be determined from milk samples stored in frozen condition or from the filters with collected cells kept at the temperature of 4 degrees C (10 days) or 25 degrees C (3 days). Milk stabilized with formaldehyde can also be used for the determination if stored at 4 degrees C.