Freezing mouse blastocysts. The influence of the preparations prior to freezing on the survival rate of the blastocysts

A good survival rate in culturing mouse blastocysts can be obtained in Ovum Culture Medium, enriched with 20 per cent inactivated Foetal Bovine Serum or Sheep Serum under air. The transfer of fresh blastocysts gives the best results if the recipients are on day 3 of the pseudo-pregnancy, but with 20 hours' cultured blastocysts it is better to use recipients on day 4. Exposure to 1.5 M DMSO has no harmful effect, provided that the DMSO is added at 5 degrees C in 6 steps and is removed, again in 6 steps, at 35 degrees C. The crystallization of the medium containing the embryos at -5 degrees C to -6 degrees C doet not appear te have a harmful influence on culture results of the blastocysts.     

The effects of temperature on the silage microbiology and aerobic stability of corn and vetch-grain silages

Abstract

The purpose of the current work was to extend the study of the effect of temperature on silage microbiology, with or without formic acid, and on the aerobic stability of corn and vetch-grain silages.

The silage samples were ensiled in 1.0-l anaerobic jars, with and without formic acid, at room (20°C) or elevated temperatures (30–37°C). After 45 days of ensiling, the silages were subjected to an aerobic stability test at room (20°C) and elevated (30–37°C) temperatures. The most intensive deterioration occurred at 30–37°C. Samples incubated at 30–37°C had the highest yeast and mould count, most prolific CO₂ production.

The finding of the current study suggests that formic acid may decrease mould growth in silage samples. Unfortunately, formic acid does not reduce aerobic deterioration rate of silages. Applying a 5 g/kg formic acid on corn and vetch-grain silages was not very effective at high temperatures.     

Comparison of storage time‐temperature effects on sensory and hedonic attributes of frozen and deep‐frozen chickens

1. Broilers were stored at ‐12±1°G and ‐18±1°C for nine periods of up to 24 and 36 months respectively and compared with birds stored at ‐43 ± 2°C.

2. There were negligible differences in preference between the experimental and reference grilled breast meats.

3. Odour preference differences for thawed, uncooked birds were significant after 1 month of storage at ‐ 12 °C and after 9 months at ‐ 18 °G.

4. In comparison with the reference birds the redness of frozen and thawed birds decreased more regularly during storage at ‐ 12°C than at ‐18 °C.

5. Packaging the birds in Cryovac instead of in polythene resulted, in the raw birds, in a greater difference in surface redness. This redness decreased more rapidly during storage than that of birds packaged in polythene.     

Intramuscular administration of alfaxalone in red‐eared sliders (Trachemys scripta elegans) – effects of dose and body temperature

ObjectiveTo characterise the effects of alfaxalone by intramuscular (IM) injection in red-eared slider turtles and the influence of body temperature on anaesthetic duration and depth.Study designProspective, randomised part-blinded experimental trial.AnimalsTen healthy adult female red-eared sliders.MethodsEach turtle was anaesthetized four times with 10 and 20 mg kg^?1 alfaxalone at 20 and 35 °C respectively. Time to maximal effect and plateau and recovery periods were recorded. Skeletal muscle tone, presence of various reflexes, response to noxious stimuli, and heart rate were assessed.ResultsResults are given for protocols 10 mg kg^?1 20 °C; 20 mg kg^?1 20 °C; 10 mg kg^?1 35 °C and 20 mg kg^?1 35 °C, respectively: mean time (±SD) to maximal effect was 16 ± 8, 19 ± 6, 5 ± 2 and 7 ± 5 minutes; duration of the plateau phase was 13 ± 12, 28 ± 13, 8 ± 5 and 8 ± 5 minutes and recovery time was 76 ± 20, 126 ± 17, 28 ± 9 and 41 ± 20 minutes. Endotracheal intubation was successful in 80%, 100%, 0% and 30% of turtles, respectively. At 35 °C, all animals retained nociceptive sensation in the front limbs, hind limbs and vent, whereas at 20 °C a few turtles lost peripheral nociceptive sensation. Corneal and tap reflexes were retained in all trials. Mean heart rates were 30 ± 2 and 66 ± 4 beats minute^?1 at 20 and 35 °C, respectively.Conclusions and clinical relevanceAlfaxalone administered IM in red-eared sliders provided smooth, rapid induction and uneventful recovery. At 35 °C either dosage provided only short (5–10 minutes) and light sedation. At 20 °C, 10 mg kg^?1 provided sedation suitable for short non-invasive procedures. About 20 mg kg^?1 provided anaesthesia of approximately 20 minutes duration, appropriate for induction of inhalational anaesthesia or for brief surgical procedures with supplemental analgesia.     

Development of an ELISA system for Detection of Anti‐Anaplasma marginale Antibodies in cattle in Brazil

An ELISA test was developed for detecting antibodies against Anaplasma marginale in bovine sera. Four antigenic preparations were produced from infected red blood cells. Some aliquots of this preparation were stored at ‐70°C with 30% DMSO in phosphate‐buffered saline (PBS) and others were lysed with 0.9% NH₄Cl and stored at ‐ 20°C. Typical anaplasmal structures were seen by electron microscopy in the antigenic preparations containing the erythrocytes that had been stored with DMSO. The performance of the ELISA test was evaluated by testing 298 positive serum samples collected from immunized cattle, 39 negative serum samples collected from cattle imported from areas free of A. marginale and 50 samples collected from cattle naturally infected in the field. The test gave a specificity of 94.87% and a sensitivity of 100%.     

In vitro reactivity to concanavalin A of bovine lymphocytes after cryopreservation

Cryopreservation of bovine peripheral lymphocytes and its effect on the $\text{in vitro}$ response to concanavalin A tested in a microculture system is described. Using DMSO as cryoprotectant in the medium, the cells were cooled to ?30°C at 1.3°C/minute and further to ?80°C at 6°C/minute and then rapidly to ?196°C by dropping in liquid nitrogen. The cells were recovered by rapid thawing in water at 30–35°C and washed twice before use in the stimulation test. Ten percent DMSO had a much better protective effect than 5%; addition of 25% fetal bovine serum to the freezing had no favourable effect. In most of the 16 animals used in the experiments the frozen lymphocytes gave the same or a higher response to Con A than those kept in the DMSO containing medium at 4°C for two hours.The responses of the frozen cells were comparable to those of fresh lymphocytes (kept at 4°C for two hours in medium without DMSO).     

The effect of processing and marketing procedures on the bacteriological condition and shelf life of eviscerated turkeys

Experiments carried out in a turkey processing plant showed that there was a fivefold increase in the number of psychrophilic bacteria present after holding eviscerated carcasses in static slush ice tanks for 24 hr. The predominant bacteria in the 24 hr chill tanks were strains of Flavobacterium, Cytophaga and Pseudomonas.

The carcasses were wrapped in heat‐shrunk‐oxygen‐impermeable film and after freezing packed in individual boxes and held at ‐20° C. The storage life at 1°, 10° and 20° C. was determined together with the time taken for the frozen carcasses to equilibrate to these temperatures. It was found that at 1° C. the carcasses kept for about 3 weeks, but at 10° C. they spoiled within 7 days and at 20° G. within 3 days.

An analysis of the spoilage flora showed that although Pseudomonas strains predominated on the turkeys stored at 1° C. fewer were isolated from turkeys stored at 10° C. and 20° C. At these two temperatures an organism identified as Enterobacter liquefaciens predominated together with atypical lactobacilli resembling unidentified strains previously described by Thornley and Sharpe (1959).     

The Effect of Temperature and Salinity on the Elimination of Enrofloxacin in the Manila Clam Ruditapes philippinarum

Abstract

The effect of temperature and salinity on the elimination of enrofloxacin (EF) in Manila clams Ruditapes philippinarum was investigated. The clams, cultured under different temperatures and salinities (16°C and 30‰, 22°C and 30‰, or 22°C and 20‰), were exposed to EF at 5 μg/mL of water in a medicated bath. After a 24-h exposure, the concentration of EF in various tissues was measured by high-performance liquid chromatography and the elimination rate of EF in those tissues was investigated by regression analysis. After the treatment, the initial concentrations of EF among tissues were (in decreasing order) plasma > gill > visceral mass > foot > adductor muscle. In all tissues the elimination half-life (t _1/2) of EF in the clams cultured at 22°C and 20‰ and 16°C and 30‰ were markedly longer than in those cultured at 22°C and 30‰, and the t _1/2 at 16°C and 30‰ was slightly longer than that at 22°C and 20‰. Slight differences were also observed in t _1/2 values among various tissues. These data indicate that both temperature and salinity had significant effects on the elimination of EF in the Manila clams and that lower temperature or salinity could result in slower elimination.

Received January 21, 2011; accepted December 2, 2011.     

Effects of ambient temperature on growth performance and carcass traits of male growing White Pekin ducks

ABSTRACT

1. An experiment was conducted to investigate the effects of ambient temperature on growth performance and carcass traits in male growing Pekin ducks from 14 to 42 d of age in order to establish their optimal temperature requirements.

2. A total of 216 14 d old male White Pekin ducks were allocated randomly to six environmentally controlled chambers with ambient temperature set at 20°C, 22°C, 24°C, 26°C, 28°C, and 30°C from 14 to 42 d of age, respectively.

3. As ambient temperature increased from 20°C to 30°C, the body weight and weight gain decreased linearly or quadratically (P < 0.05) and was accompanied by linearly decreasing feed intake (P < 0.05). According to broken-line regression, the upper critical level of ambient temperature during the growing period for body weight, weight gain, and feed conversion ratio were 27.4°C, 27.4°C, and 26.0°C, respectively.

4. The weight of breast meat, leg meat, and abdominal fat decreased linearly or quadratically as ambient temperature increased and declined to a minimum when the temperature increased to 30°C (P < 0.05). The percentage of breast meat and abdominal fat showed a linear or quadratic decreasing response to increasing temperature, but leg meat percentage increased as temperature increased and reached maximum at 30°C (P < 0.05). According to broken-line regression, the upper critical ambient temperatures during the growing period for breast meat weight and percentage were 25.5°C and 25.6°C, respectively.

5. It was concluded that both growth performance and breast meat of growing ducks were sensitive to increasing ambient temperature and this should be kept below the upper critical temperature during the growing period in order to optimise growth performance and carcass traits at market age.     

The effect on creatinine kinase activity of freezing plasma from healthy foals

Abstract

AIM: To assess the stability of creatine kinase (CK) activity in plasma collected from healthy foals and frozen at ?20°C for up to 12 weeks.

METHODS: Samples of venous blood drawn from 25 foals were analysed for CK activity soon after collection, and again after 1 and 12 weeks of freezing at ?20°C.

RESULTS: CK activity decreased (p<0.001) between Week 0 and Week 1 and between Week 0 and Week 12.

CONCLUSIONS AND CLINICAL RELEVANCE: Decreases in CK activity were statistically significant but clinically insignificant.     

Comparison of Two Biochemical Test Systems with Conventional Methods for the Identification of Bacteria Pathogenic to Warmwater Fish

Abstract

One hundred seven Aeromonas spp., 26 Edwardsiella ictaluri, 6 E. tarda, 12 Plesiomonas shigelloides, and 6 Pseudomonas spp. (131 piscine isolates and 26 reference isolates) were studied with 36 biochemical tests from the Minitek system, 20 tests from the API 20E system, and corresponding standard tube tests. Isolates were incubated at 25°C. Arginine dihydrolase, ornithine decarboxylase, mannose, and citrate showed less than 95% agreement between the Minitek system and the tube tests. Arginine dihydrolase, lysine decarboxylase, nitrite reductase, Voges-Proskauer, and citrate showed less than 95% agreement between the API 20E system and the tube tests. The 26 reference isolates were examined with the three systems and were incubated at both 25 and 37°C. There were no major differences between tests run at 25 and 37°C except with nine Aeromonas spp. that did not grow well at 37°C. Both the Minitek and API 20E systems will reproduce standard biochemical tube test results with at least 95% accuracy when used to test warmwater fish pathogens incubated at 25°C. However, the numerical identification databases for both the Minitek and API 20E systems were not usable for identifying fish pathogens.     

Extending the viability of emu spermatozoa during in vitro storage by manipulation of temperature and diluent potassium concentration

1. Survival of emu spermatozoa during in vitro storage is not affected by increasing the extracellular [K⁺] to the point where it does not adversely affect spermatozoa function.

2. In three experiments, the effects were studied of [K⁺] in a diluent in the range 12·5–80?mM/l on emu spermatozoa survival for up to 48?h at 5, 10 or 20°C.

3. At the end of the storage period, spermatozoa viability, motility, fertilising ability and morphology were measured.

4. In Experiment 1, spermatozoa viability and morphology were adversely affected after storage (P?M/l [K⁺] whereas spermatozoa motility decreased as [K⁺] increased from 12·5 to 80?mM/l.

5. In Experiment 2, during storage at 5°C, the spermatozoa viability was comparable among any of the diluents (standard or modified) but morphology was better (P? 6. In Experiment 3, after 48?h of storage in a diluent containing 40?mM/l of [K⁺], the spermatozoa functions were better preserved at 10°C than at 5 or 20°C.

7. It is concluded that a higher than physiological level of potassium can be used in a diluent without detrimental effect on emu spermatozoa survival during 48?h storage and that the best outcome was with storage at 10°C rather than 5 or 20°C.     

Effects of Water Temperature on Experimental Transmission of Dermal Sarcoma in Fingerling Walleyes

Abstract

The effects of temperature on experimental transmission of dermal sarcoma, a spontaneous tumor of adult walleyes Stizostedion vitreum to fingerling walleyes was determined. Test temperatures were 10, 15, and 20°C. Three-month-old walleyes were inoculated intramuscularly with a cell-free filtrate from a dermal sarcoma collected from an adult walleye in the spring of the year. Tumors were first grossly visible in fish held at 15°C at 8 weeks postinoculation. At 12 weeks postinoculation, all fish were euthanized and examined for presence of tumors. Tumor transmission was most successful at 15°C, followed by that at 20°C; many fish maintained at these temperatures had grossly visible tumors. Although the majority offish held at 10°C also had tumors, tumors in these fish developed to a lesser degree than those observed in fish held at the higher temperatures.     

Pharmacokinetics of sulphadimidine in carp (Cyprinus carpio L.) and rainbow trout (Salmo gairdneri Richardson) acclimated at two different temperature levels

Summary

The influence of temperature (10° C and 20° C) on pharmacokinetics and metabolism of sulphadimidine (SDM) in carp and trout was studied.

At 20° C a significantly lower level of distribution (Vd_area ) and a significantly shorter elimination half‐life (T _(½>) β) was achieved in both species compared to the 10° C level. In carp the body clearance parameter (Cl_B (SDM) was significantly higher at 20° C compared to the value at 10° C, whereas for trout this parameter was in the same order of magnitude for both temperatures.

N₄‐acetylsulphadimidine (N₄‐SDM) was the main metabolite of SDM in both species at the two temperature levels. The relative N₄‐SDM plasma percentage in carp was significantly higher at 20° C than at 10° C, whereas there was in trout no significant difference.

In neither species was the peak plasma concentration of N_4‐SDM (C_maxN4‐SDM)) significantly different at two temperatures.

The corresponding peak time of this metabolite (T_{max (N4‐SDM)}) was significantly shorter at 20° C compared to 10° C in both carp and trout.

In carp at both temperatures, acetylation occurs to a greater extent than hydroxylation. Only the 6‐hydroxymethyl‐metabolite (SCH₂OH) was detected in carp, at a significant different level at the two temperatures. Concentrations of hydroxy metabolites in trout were at the detection level of the HPLC‐method (0.02‐μg/ml). The glucuronide metabolite (SOH‐gluc.) was not detected in either species at the two temperatures.     

The effect of storage temperature on the shelf‐life of eviscerated air‐chilled Turkeys

1. Eviscerated air‐chilled turkeys (weighing about 5.5 kg) were stored in groups of 10 at temperatures between 5 and — 2 °C. Slight “off” odour was detected in an average time of 7.2 d at 5 °C, 13.9 d at 2 °C, 22.6 d at 0 °G and about 38 d at ‐2 °C.

2. The microbiological condition of the carcasses was determined initially and after storage at — 2 ^oC for 28, 35 and 42 d. It was found that, whilst pseudomonads (pigmented and non‐pigmented) were present at 10⁸/cm² after 35 and 42‐d storage, yeasts were also present at 10⁷/cm² and probably accounted for the unusual fusty “off” odours.     

Oxidative stability and α‐tocopherol retention in turkey burgers during refrigerated and frozen storage as influenced by dietary α‐tocopheryl acetate

1. The effect of vitamin E (α‐tocopheryl acetate) in turkey diets on the oxidative stability of raw and cooked turkey burgers and on the retention of α‐tocopherol during refrigerated (4°C) or frozen (‐20°C) storage was investigated. One hundred and two, one‐day‐old T‐8^S turkey poults were divided at random into 3 groups of 34 animals each and fed on either a basal diet (normal commercial turkey diet) supplemented with 20 mg α‐tocopheryl acetate/kg (control) or fed an α‐tocopherol supplemented diet containing 300 (E300) or 600 (E600) mg α‐tocopheryl acetate/kg for 21 weeks.

2. Dietary supplementation with α‐tocopheryl acetate significantly reduced TBARS numbers in both raw and cooked burgers during refrigerated and frozen storage.

3. The mean values of α‐tocopherol in raw and cooked burgers stored at 4°C did not change during storage.

4. In the case of both raw and cooked samples stored at ‐20°C, the α‐tocopherol values decreased from 5.67 to 3.54 and from 3.56 to 2.30 μg/g in the raw burgers from turkeys from the E600 and E300 treatments, respectively, after 4 months storage. The values decreased from 5.60 to 2.88 and from 3.29 to 1.85 μg/g in cooked burgers from turkeys from the E600 and E300 treatments, respectively, after 5 months storage.     

The temperature of storage of a batch of wheat distillers dried grains with solubles samples on their nutritive value for broilers

1. A batch of wheat distillers dried grains with solubles (DDGS) was obtained immediately after production and was separated into 5 equal parts and placed in woven polypropylene sacks. The samples were stored under 5 different temperature conditions for 1 year as follows: kept at a constant ?20°C; kept at ?20°C for 24 h period and after that kept at a constant +4°C; kept at a constant +4°C only; kept at a constant +15°C; stored at ambient temperature (range of weekly mean temperatures was from +4 to +22°C).

2. Each of the 5 wheat DDGS samples was included (200 g/kg) in a nutritionally complete diet and fed to broiler chickens from 7 to 21 d of age. The chemical composition of the DDGS samples was determined at the beginning and at the end of the 1-year storage period.

3. The nitrogen corrected apparent metabolisable energy (AMEn) and the nutrient availability of each sample was measured using a total collection technique. The growth performance of birds was also determined.

4. The DDGS samples kept at a constant ?20°C had higher dry matter, lower oxidation value and lower antioxidant contents. The DDGS sample that was stored at ambient temperatures had a higher AMEn than the rest of the DDGS samples.

5. The results of this experiment have shown that there can be changes in the AMEn of wheat DDGS during storage at ambient temperatures. In general, there were no serious effects of storage of DDGS on its feeding value to broiler chickens.     

Effect of different penetrating and non‐penetrating cryoprotectants and media temperature on the cryosurvival of vitrified in vitro produced porcine blastocysts

The aim of this study was to determine the most efficient vitrification protocol for the cryopreservation of day 7 in vitro produced (IVP) porcine blastocysts. The post‐warm survival rate of blastocysts vitrified in control (17% dimethyl sulfoxide + 17% ethylene glycol [EG] + 0.4 mol/L sucrose) and commercial media did not differ, nor did the post‐warm survival rate of blastocysts vitrified in medium containing 1,2‐propandiol in place of EG. However, vitrifying embryos in EG alone decreased the cryosurvival rate (55.6% and 33.6%, respectively, p < .05). Furthermore, the post‐warm survival rates of blastocysts vitrified with either trehalose or sucrose as the non‐penetrating cryoprotectant did not differ. There was also no significant difference in post‐warm survival of blastocysts vitrified in control (38°C) media and room temperature (22°C) media with extended equilibration times, although when blastocysts were vitrified using control media at room temperature, the post‐warm survival rate increased (56.8%, 57.3%, 72.5%, respectively, p < .05). The findings show that most cryoprotectant combinations examined proved equally effective at supporting the post‐warm survival of IVP porcine blastocysts. The improved post‐warm survival rate of blastocysts vitrified using media held at room temperature suggests that the cryoprotectant toxicity exerted in 22°C media was reduced.     

Effective,appropriate and simple culture,egg hatching and cryopreserving of the nematode Cheilospirura hamulosa

1. Successful invasion by nematode parasites is associated with several factors including egg hatching at the right time in their hosts. To determine a simple and appropriate medium for culture and egg hatching of the highly pathogenic species of the Acuariidae family, Cheilospirura hamulosa were cultured in three different media. In addition the viability of C. hamulosa eggs was determined after storage in frozen infected gizzards.

2. Eggs removed from the uteri of the female worms in infected gizzards were pooled and washed in distilled water and screened under a stereo dissecting microscope. Eggs were counted and cultured in three different media, nutrient agar, normal saline 0.9% and Bearman, at room temperature. Additionally, 10 infected gizzards were kept at ?20°C for 2 and 8 months.

3. After 4–5 d there had been no growth in the nutrient agar medium, whereas 11% of the cultured eggs in the Bearman medium contained larvae 2–3 d after culturing. In 0.9% normal saline medium the two polar knobs appeared on the two poles of the eggs at 2 d post cultivation, and 74% of the eggs contained a larva on the third day. Mature larvae gradually exited from the eggs.

4. Eggs collected from female worms in gizzards frozen at ?20°C were cultured in the same three culture media at room temperature. Larvae were visible in the eggs after 2–3 d in the Bearman and 0.9% normal saline media and hatched thereafter.

5. The 0.9% normal saline medium is recommended for egg hatching and cultivation of C. hamulosa due for simplicity, efficacy and cost effectiveness. Moreover, freezing of the infected gizzards at ?20°C is proposed for long-term storage of the eggs.