The effect of urine storage temperature and boric acid preservation on quantitative bacterial culture for diagnosing canine urinary tract infection.

BACKGROUND: Quantitative bacterial culture (QBC) is the gold standard for diagnosing canine urinary tract infection. Current guidelines recommend QBC within 24 h of urine collection and that unpreserved urine is refrigerated until culture. However, temperature-controlled transport is rarely feasible, indicating a need for alternative storage during transport of urine from primary veterinary practices to the microbiology laboratory. The objective was to investigate the effect of storage temperature and boric acid sponge-preservation on quantitative bacterial culture of canine urine. RESULTS: Significant bacteriuria was detected in 72 out of 179 samples (40%) collected from 141 dogs. Overall accuracy was 94-98% for both storage conditions and time points. Non-inferiority (15% margin) to reference quantitative bacterial culture was evident for sensitivity, specificity and predictive values for both storage methods and time points, except for the negative predictive value for 48 h boric acid preservation (NPV: 89, 95% CI [79;95]). There was no significant difference between the sensitivity and specificity for either of the time-points (p-value = 0.07-1). CONCLUSIONS: Boric acid sponge-preservation using Uriswab™ is a useful alternative to refrigeration of urine samples during transport. Reliable quantitative bacterial culture results can be obtained from canine urine up to 48 h after collection if urine is refrigerated, and for at least 24 h if urine is stored using a boric acid-containing urine transport system.

Influences of swab types and storage temperatures on isolation and molecular detection of Mycoplasma gallisepticum and Mycoplasma synoviae.

Routine diagnosis of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) is performed by collecting oropharyngeal swabs, followed by isolation and/or detection by molecular methods. The storage temperature, storage duration and the type of swab could be critical factors for successful isolation or molecular detection. The aim of this study was to compare the influence of different types of cotton-tipped swab stored at different temperatures, on the detection of MG and MS. To achieve this, combined use of traditional culture analysis (both agar and broth), with modern molecular detection methods was utilized. Performances of wooden and plastic shaft swabs, both without transport medium, were compared. Successful culture of M. gallisepticum was significantly more efficient from plastic swabs when compared to wooden, whereas no difference was seen for the re-isolation of M. synoviae. Storage at 4°C compared to room temperature also increased the efficiency of culture detection for both Mycoplasma species. When stored at room temperature, PCR detection limits of both MG and MS were significantly lower for wooden compared to plastic swabs. The qPCR data showed similar detection limits for both swab types when stored at both temperatures. The results suggest that swabs with a plastic shaft are preferred for MG and MS detection by both culture and PCR. While a lower storage temperature (4°C) is optimal for culture recovery, it seems that both temperatures investigated here are adequate for molecular detection and it is the swab type which carries a greater influence.

Accuracy of body mass estimates of formalin-preserved fish - a review.

This paper highlights possible effects of physical and chemical mechanisms of formalin fixation and preservation on biological tissue and reviews the consequent potential inaccuracies on estimates of body mass of small fishes fixed and preserved in formalin. Twenty-six papers including 65 independent experiments with 35 species which examine effects of formalin on body mass estimates on small fishes are included. The effect of the formalin on the specimens depends on the salinity of the water used to dilute the commercial formalin (usually 1:9 formalin: water) before being used to fixate and preserve fish. Mean wet body mass of the specimens from the studies using seawater or fresh water diluted formalin deceases by 13% and increases by 7%, respectively, from before to after being immersed in formalin. The same trend is found with condition factor in the few papers that report this parameter. Body length decreases on average by c. 2% in fixated and preserved fish regardless of whether the formalin is diluted in seawater or fresh water.

Impact on canine neutrophil preservation with the addition of bovine serum albumin to K3 -EDTA whole blood samples.

BACKGROUND: Cellular deterioration occurs with blood sample aging, impacting white blood cell (WBC) identification and differential accuracy. This may be exacerbated in samples from patients experiencing inflammation. Previously, bovine serum albumin (BSA) has been shown to improve cellular preservation of blood and other samples, but the effect on cell preservation in canine blood has not been assessed. OBJECTIVES: We aimed to determine the effects of BSA on neutrophil nuclear area when added to potassium ethylene diamine tetra-acetic acid (K3 -EDTA)-anticoagulated canine blood prior to blood smear preparation. We evaluated the impact of inflammatory leukograms, sample storage temperatures (4° and 20°C), and time on outcomes. MATERIALS AND METHODS: Canine K3 -EDTA-anticoagulated blood samples stored at 4° and 20°C were used from unique patients, 10 with and 10 without inflammatory leukograms. Blood smears were prepared from aliquots with or without the addition of 22% BSA at 0, 4, 8, 24, 48, and 72 h. The nuclear area was measured for 25 randomly selected neutrophils per slide using Fiji software. Mixed-effect linear regression modeling was performed (significance: P < 0.05). RESULTS: Nuclear area increased over time with and without added BSA. Both sample storage temperatures and the presence or absence of an inflammatory leukogram significantly impacted neutrophil nuclear area. Samples with added BSA had slightly higher predicted nuclear areas than those without BSA, but this difference was not statistically significant. CONCLUSIONS: BSA did not significantly impact neutrophil nuclear area and did not improve neutrophil preservation in canine blood samples.

Swine viscera preservation in hypersaturated salt solution after alcohol fixation as a preparation method for educational purposes.

The use of live animals for educational purposes is an old practice that is still employed in teaching and research institutions. However, there are several objections to this practice, whether for ethical or humanitarian reasons. Surgical techniques teaching using anatomical pieces and/or preserved cadavers promotes greater learning efficiency, provides exercise repetition and increases the confidence and satisfaction of the students when compared to the use of live animals. The current work aimed to analyse the feasibility of using fresh swine urinary bladder and small intestines (jejunum), obtained from slaughterhouses, fixed in 99.8% ethyl alcohol (EA) and preserved in sodium chloride hypersaturated solution (SCHS) at 30%, for 7, 14 and 21 days, as an alternative method for surgical skills training (SST). Swine viscera, fixed in EA and preserved in SCHS, presented a realistic appearance, absence of odour and maintained the viable morphological characteristics during the performance of the operative techniques. Preservation solutions had low cost, were easy to acquire and did not offers risks to human health. Therefore, urinary bladders and small intestines fixed in 99.8% EA for 30 days and maintained in 30% SCHS at different periods were demonstrated as a good viable option as a preservation method for surgical skills training.

Preservation of viable Taylorella equigenitalis in different commercially available transport systems.

The cultural diagnosis of the causal agent of contagious equine metritis (Taylorella equigenitalis) using transport swabs is challenging. Swabs must be placed in Amies charcoal medium, refrigerated during transport, and plated out at the laboratory no later than 48 h after sampling. In this study, the viability of T. equigenitalis strain CIP 79.7T in 11 commercial swab transport systems was initially compared at 1 day and 2 days of storage at ambient (20 ± 3 °C) or refrigerated (5 ± 3 °C) temperature. The four best swab transport systems, systems B, E, F (used as the reference) and K, were then compared at 0, 2, 3, 4, 7 and 10 days at refrigerated temperatures. Statistically significant differences were observed after 10 days only for system K compared to the reference, with approximately 95% viable T. equigenitalis recovered in system K compared to approximately 77% in system F. System K is thus promising for preservation and transport of viable T. equigenitalis for culture.

The use of cryomicroscopy in guppy sperm freezing.

The present study employed cryomicroscopy to derive an optimal sperm freezing protocol for guppy (Poecilia reticulata) sperm. Evaluation criteria during the freezing-thawing process were assessed for nucleation temperature (Tn), temperature when more than 50% of sperm display bending mid-piece (Tb), temperature when more than 80% of sperm stop moving (Tm), thawing temperature (Tt), and post-thaw motility. We compared four different cryoprotectants: 5% N-dimethyl formamide (DMF), 6% methanol (MEOH), 10% dimethyl sulfoxide (DMSO), and 14% glycerol, as well as glycerol at different concentrations of 7-50%; cooling and rewarming rates ranged from 5 to 100°C/min. The protocol that yielded the highest post-thaw motility was samples suspended in 14% glycerol, cooled at 25°C/min, and thawed at 100°C/min, which was in complete agreement with our previous findings derived from a controlled-rate freezer. In addition, Tb and Tm were found to be negatively correlated with post-thaw motility, suggesting their possible role in predicting freezing success. The present study for the first time demonstrated the usefulness of cryomicroscopy in deriving an optimal sperm freezing protocol for aquatic species.

Effective nitrogen preservation during urine collection from Holstein heifers fed diets with high or low protein content.

Six Holstein heifers (body weight=535-625 kg) fed a total mixed ration containing either high protein (13.4%) or low protein (9.0%) were used to evaluate the effect of 3 urine collection methods (chilled, acidified before collection, or acidified after 6h of collection) on urinary N preservation. In a 2-period crossover design, 16-d diet adjustment stages preceded five 24-h collections. Urinary catheters were inserted 1 d before the collection periods. Urine collection tubes were configured to split urine to 3 collection containers: 1 acidified with 6 N HCl before collection at a rate calculated to reduce pH to below 2, 1 acidified every 6h during collection to pH below 2, and 1 located in a large cooler of ice. Collection method did not affect urinary concentration of N or urine urea-N (9.2+/-0.9 g/L and 6.58+/-0.9 g/L, respectively) or urinary excretion of N or urea-N (82+/-3.8 g/d and 59.5+/-3.8 g/d, respectively). These 3 collection methods are equally effective in preserving N during urine collection, but the "chilled immediately" approach may be useful for studies focused on ammonia emission. Urinary and fecal N excretion were significantly different across collection days; fecal N was more highly variable than urinary N. Intake and apparent N digestibility decreased during the collection week, and excretion of urinary and fecal N increased, particularly on d 5. (Stable rectal temperatures suggested no urinary infections.) Improvements in total collection methodology will support continued progress in the understanding of livestock N utilization and post-excretion changes in manure N.

Effect of storage media, temperature, and time on preservation of Cryptosporidium parvum oocysts for PCR analysis.

The effect of storage media, temperature, and time on suitability of oocysts for use in subsequent molecular studies was examined. Cryptosporidium parvum oocysts were stored for 3, 6, 9, or 12 months in sterile dH(2)O, 70 or 95% ethanol, (room temperature [RT], 4, -20, and -70 degrees C), 10% formalin (RT and 4 degrees C), PBS, TE buffer, antibiotic-antimycotic (A-A) solution (4, -20 and -70 degrees C), 2% sulphuric acid, 2.5% potassium dichromate (4 degrees C), and gDNA from 10(4) oocysts was extracted in triplicate and subjected to PCR. To determine the effect of storage media on PCR sensitivity, gDNA from 10(4), 10(2), and 10(0) oocysts stored for 15 months in the media listed above at RT or 4 degrees C was also extracted in triplicate and subjected to PCR. At RT, ethanol was suitable for up to 15 months, while gDNA from oocysts stored in dH(2)O amplified inconsistently after 3 months. At 4 degrees C, all tested media except dH(2)O and formalin were suitable for storage of 10(4) oocysts up to 15 months, but only 70% ethanol, A-A solution, 2% sulphuric acid and 2.5% potassium dichromate supported amplification of gDNA from fewer than 100 oocysts. At -20 degrees C, 95% ethanol, PBS, or TE were suitable for up to 9 months, while 70% ethanol and A-A solution were effective up to 12 months, and gDNA from oocysts stored in dH(2)O was inconsistently amplified after 6 months. Storage at -70 degrees C for up to 12 months was effective regardless of media type. Oocysts stored in formalin at RT or 4 degrees C could not be amplified by PCR despite washing prior to gDNA extraction. To maintain gDNA quality suitable for PCR, it is recommended that coccidian oocysts be stored at -70 degrees C in dH(2)O, ethanol, PBS, TE or A-A solution, at 4 degrees C in A-A or ethanol, or at RT in ethanol where refrigerated storage is unavailable.

Vitrification of early-stage bovine and equine embryos.

The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5M ethylene glycol (EG) for 5min, 7M ethylene glycol and 0.6M galactose for 30s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1M ethylene glycol and 1.1M dimethyl sulfoxide (DMSO) for 3min, 2.5M ethylene glycol, 2.5M DMSO and 0.5M galactose for 30s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P>0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (P<0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher (P<0.05) for EG-O than EG-DMSO. Therefore, EG-O was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1-3 d. Two- to eight-cell embryos were vitrified, warmed and transferred into recipient's oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI.

Exploring dry storage as an alternative biobanking strategy inspired by Nature.

Biobanking is a rapidly growing industry, covering diverse fields such as human medicine, farm animal production, laboratory animals record keeping, and wildlife conservation. Presently, biobanking is done almost exclusively by cryopreservation, followed by maintenance of the samples under liquid nitrogen. Cryopreservation has satisfactory efficiency but it comes with a host of problems, and the process is highly species-specific. Like in many other walks of life, we turn to Nature in search for better alternatives. Nature opted for controlled drying rather than water preservation via freezing when long-term preservation is desired, a strategy known as 'anhydrobiosis'. To achieve reversible drying, anhydrobiotic organisms utilise an assortment of protective materials, including disaccharides, late embryogenesis abundant proteins, anhydrin, heat shock protein, and more. Once dry, desiccation-tolerant organisms can survive extended periods of time and be resistant to extreme environmental stressors. Over the past 70 years researchers attempted applying this idea to preserve desiccation-sensitive mammalian cells in the dry form. At present dried cells mostly do not resume biological activity upon rehydration. The DNA, however, is often well preserved to allow utilisation in advanced reproductive techniques. Spermatozoa are by far the most commonly dried cell type, primarily from mice and bulls. A number of drying approaches have been applied, with freeze-drying taking the lead. To date offspring have been produced from dried spermatozoa in mouse, rat, hamster, rabbit, and horse. No offspring were produced from dried somatic cells. Desiccation experiences a sharp increase in interest and research output in recent years. Presented here is an overview of dry preservation, its possible applications, the open questions the field is still facing, and some suggested directions for the future.

Evaluation of fecal microRNA stability in healthy cats.

BACKGROUND: Gastrointestinal (GI) cancer accounts for 14% of feline malignancies. There is a great need for reliable noninvasive diagnostic biomarkers to reach a timely diagnosis and initiate treatment. Fecal microRNAs (miRNAs) could be such a biomarker and have shown great potential in colorectal screening in people but have yet to be investigated in cats. OBJECTIVES: We aimed to evaluate the presence and stability of feline fecal miRNA under different storage conditions (room temperature [RT], 4, and -20°C) and to evaluate the expression levels of specific fecal miRNAs collected on three separate days (days 1, 4, and 7) in healthy cats. METHODS: Healthy cats were prospectively recruited. Fecal samples were collected, aliquoted, and stored for 24 hours at RT and then transferred to -20°C, stored for 24 hours at 4°C and then transferred to -20°C, or were immediately placed at -20°C on day 1 or at -20°C on days 4 and 7 postcollection. Expression of 22 miRNAs was investigated using quantitative real-time PCR. RESULTS: Ten miRNA assays worked well, and one, let-7b, was used for normalization. No differences in miRNA expression were seen between the three storage temperatures for the nine miRNAs investigated. Only miR-26a showed a significant increase in expression between samples of days 1 and 7. The rest of the miRNAs levels were stable over time. CONCLUSIONS: Fecal miRNA can be isolated from healthy cats. The expression was stable at different temperatures and for most of the miRNAs over time. Prospective studies evaluating fecal miRNA as biomarkers in cats with GI neoplasia are warranted.

Strategies for the storage of Ancylostoma caninum third-stage larvae.

Although cryopreservation protocols for storage of hookworm larvae have been described, the circumstances under which the technique is necessary to ensure larval survival are not well defined. The motility of infective-stage larvae (as judged by observation) and their ability to migrate through canine skin in vitro were measured over a 7-mo period in worms held at room temperature and worms that had been cryopreserved at the start of the experiment. Cryopreserved worms showed motility and migration proportions of 45.6-48.0% and 26.8- 34.0%, respectively, throughout the experiment, compared with percentages of 92.7 and 84.1%, respectively, in the original fresh worms. Larvae held at room temperature showed a gradual decrease in motility and migration ability over the experimental period. Motility and migratory ability of cryopreserved larvae was only significantly higher (P < 0.01) than room temperature-stored larvae from 4 and 5 mo onward, respectively.

Hypothermic storage of equine isolated hepatocytes.

The aim of the study was to establish the optimal methods for hypothermic storage of equine isolated hepatocytes. Viability of equine isolated hepatocytes after hypothermic storage was dependent on the type of storage medium as well as on the cell density in the storage suspension and the preservation period. Hepatocytes stored at 4 degrees C in Hanks' Balanced Salt Solution (HBSS) and Williams' Medium E (WE) for 24 h showed very low viability, numerous cell membrane blebs, very low attachment rate (11.9 +/- 6.5% and 34.8 +/- 19.1%, respectively) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction rate (6.4 +/- 3.9% and 25.1 +/- 14.8%, respectively). In contrast, hepatocytes stored in University of Wisconsin Solution (UW) after 24 h of storage at a density of 12.5 x 10(6) cells/ml showed high viability (over 70%), typical and intact morphology, high cell attachment rates and MTT reduction. Our findings clearly demonstrate that UW is a good preservation solution for equine isolated hepatocytes. Hepatocytes harvested from slaughterhouse organs can be stored at 4 degrees C in UW at a density of 12.5 x 10(6) cells/ml for at least 24 h without significant decrease in functional integrity.

Evaluation of an alternative technique for preserving crustaceans in dry conditions with joint mobility: a proposal for didactic purposes

Although crustaceans are traditionally preserved in liquids (formaldehyde and/or ethyl alcohol), those substances tend to alter their morphological aspects. Glycerin, used in human anatomy, is considered a good substitute for formaldehyde, as it preserves animals in states similar to in vivo conditions. There are no records in the literature, however, concerning the use of glycerin for conserving invertebrates. The objective of this work was to elaborate and evaluate alternative techniques for conserving the crustacean Ucides cordatus (Linnaeus, 1763). Six fixatives (1, 3, 4 and 5% formaldehyde, 70% alcohol, and dietrich solution) and two controls (positive and negative) were tested, as well as the effects of freezing before fixation on the integrity of U. cordatus specimens. Our results were evaluated with respect to nine variables. The treatments that demonstrated the best aesthetic results were 4% formaldehyde and 70% ethyl alcohol. The freezing of the animals resulted in brittle organs in all treatments tested. The technique discussed here is extremely promising for the conservation of animals for educational purposes, as it produces preserved specimens that are aesthetically similar to their in vivo conditions.

Evaluating preservation methods for identifying Anopheles gambiae s.s. and Anopheles arabiensis complex mosquitoes species using near infra-red spectroscopy.

BACKGROUND: Near-infrared spectroscopy (NIRS) has been successfully used on fresh and RNAlater-preserved members of the Anopheles gambiae complex to identify sibling species and age. No preservation methods other than using RNAlater have been tested to preserve mosquitoes for species identification using NIRS. However, RNAlater is not the most practical preservative for field settings because it is expensive, requires basic laboratory conditions for storage and is not widely available in sub-Saharan Africa. The aim of this study was to test several cheaper and more field-friendly preservation methods for identifying sibling species of the An. gambiae complex using NIRS. METHODS: In this study we describe the use of NIRS to identify sibling species of preserved An. gambiae s. s. and An. arabiensis. Mosquitoes of each species were placed in sample tubes and preserved using one of the following preservation methods: (i) refrigeration at 4°C, (ii) freezing at -20°C, (iii) drying over a silica-gel desiccant, (iv) submersion in RNAlater at room temperature, (v) submersion in RNAlater at 4°C, and (vi) submersion in RNAlater at -20°C. Mosquitoes were preserved for 1, 4, 10, 32 or 50 weeks before they were scanned. RESULTS: Storage at 4°C was the only preservation method that, up to 32 weeks, did not result in significantly lower predicted values than those obtained from fresh insects. After 50 weeks, however, refrigerated samples did not give meaningful results. When storing for 50 weeks, desiccating samples over silica gel was the best preservation method, with a partial least squares regression cross-validation of >80%. Predictive data values were analyzed using a generalized linear model. CONCLUSION: NIRS can be used to identify species of desiccated Anopheles gambiae s.s. and Anopheles arabiensis for up to 50 weeks of storage with more than 80% accuracy.

The effect of ex vivo refrigerated storage and cell preservation solution (Cyto-Chex II) on CD11b expression and oxidative burst activity of dog neutrophils.

The expression of CD11b and oxidative burst activity of dog neutrophils undergoing ex vivo refrigerated storage was studied using flow-cytometry . Additionally, the effect of a proprietary cell stabilization reagent (Cyto-Chex) on the expression of CD11b and oxidative burst activity was studied. Expression of CD11b was very high (>90% positive) on dog neutr ophils isolated from peripheral blood. Dog neutrophils showed a rapid and sustained increase in CD11b antigen density (P<0.01) during refrigerated storage, this increase was prevented by treatment with Cyto-Chex but was not completely blocked on the first day. There were no significant differences in mean antigen density between any days in the non-preserved group or between Days 1 to 4 in the Cyto-Chex treated group. The non-treated group showed significantly greater mean antigen density at all time points when compared to the preservative treated group (P<0.0001). Treatment with Cyto-Chex did not interfere with measurement of oxidative burst function on the first 2 days. Alterations of both resting oxidative activity and stimulated response were observed over time in both treated and untreated blood samples. Cyto-Chex treated samples showed a dramatic, significant decline in stimulated response after the third day of storage (P<0.001), while non-treated cells showed steadily increasing, but non-significant differences in stimulated response. Cyto-Chex was demonstrated to be a useful reagent for stabilization of dog neutrophil membrane antigens during storage, however this reagent is not recommended for preservation of cells for functional assays.

Viability and virulence of Babesia rodhaini eight years after cryogenic preservation with dimethyl sulfoxide.

Mouse blood infected with Babesia rodhaini and containing an equal volume of 4 M dimethyl sulfoxide was infective after storage at -196 degrees C for 8 years. The Babesia organisms were still able to cause lethal infections after prolonged low temperature storage.

Loss of cell-associated Marek's disease vaccine titer during thawing, reconstitution, and use.

Tests of the effects of mismanagement practices observed in hatcheries showed that very subtle deviations from the explicit directions accompanying Marek's vaccine can cause dramatic losses of vaccine potency. Such mismanagement was found to result in 14 to 97% loss of vaccine titer. It appears that procedures used in hatcheries for the handling of cell-associated Marek's vaccine should be closely monitored on a continuous basis to prevent a gradual drift from correct handling.

Correlation of titer, preservation method, and storage of Mycoplasma gallisepticum F strain and the immune response in chickens.

The F strain of Mycoplasma gallisepticum (MG) was used either fresh or after lyophilization or freezing at -60 C to vaccinate young leghorn chickens. Vaccine stored either frozen or lyophilized for 22 months was also used. Each vaccine preparation was given at dosages ranging from 10(5) to 10(9) colony-forming units/ml. All dosage levels of MG significantly (P less than 0.05) reduced air-sac lesion scores after aerosol challenge with the R strain of MG at 6 weeks postvaccination, regardless of the method of vaccine preservation or storage.