Aversion to gaseous euthanasia agents in rats and mice

Despite euthanasia being the most common of all procedures carried out on laboratory animals, the potential distress associated with gaseous agents has received little interest until recently, with growing concern over use of carbon dioxide as a humane method of euthanasia. The distress associated with exposure to carbon dioxide, argon, and carbon dioxide-argon mixtures was investigated in rats and mice by measuring the degree of aversion on exposure to low, medium, and high concentrations of these agents. Animals were exposed to the various concentrations in a test chamber containing air or gas mixtures that they were able to enter and leave at will. Aversion was assessed, using measurements of initial withdrawal time and total dwelling time in the test chamber, as they were the most sensitive measurements of aversion. Comparisons between euthanasia agent and control (air) treatments indicated that concentrations of agents recommended for rapid and efficient induction are associated with some degree of aversion. Carbon dioxide and the carbon dioxide-argon mixtures were more aversive than was argon for rats and mice. These findings suggest that induction with carbon dioxide either alone or in combination with argon is likely to cause considerable distress before the loss of consciousness in rodents, which is unacceptable considering that effective and more humane alternatives are available.     

Behavioural responses of rats to gradual-fill carbon dioxide euthanasia and reduced oxygen concentrations

Carbon dioxide (CO₂) is widely used for euthanasia of laboratory rats, but little is known about the pain and distress caused by this procedure. Physiological and human self-report data suggest that CO₂ may cause pain and dyspnea, a sensation of breathlessness and increased respiratory effort, at the concentrations used for gradual-fill euthanasia. However, previous studies examining the behavioural responses of rats have reported conflicting results. In this study, detailed behavioural responses of rats were examined during gradual-fill CO₂ euthanasia and during gradual-fill with argon to produce a similar reduction in oxygen concentration. Animals were randomly allocated to the CO₂ or reduced oxygen treatment groups (n = 8 for both), and were first tested with air exposure and then with treatment gas exposure on the following day. Observations were taken for 105 s before and after gas flow began (baseline and exposure periods), as animals had ceased purposeful movement within 105 s of CO₂ flow starting. Behavioural changes from baseline during gas exposure were compared to changes during air exposure on control days. In comparison to air exposure, CO₂ resulted in increased activity, rearing, touching the nose to the chamber lid, escape behaviours and vocalizations. A small increase in touching the nose to the chamber lid was observed when oxygen concentrations were reduced with argon, but no other behavioural changes were observed. These results suggest that gradual-fill CO₂ euthanasia causes distress in rats, and that hypoxia alone is not a major cause of this distress.     

A potent hypotensive factor in chicken left ventricle

Chicken artria and ventricles both have membrane-bound granules which resemble those containing atriopeptin (ANP) in mammals. However, nothing is known about the contents of the avian granules. A previous study in chickens showed that although extracts of whole chicken heart or synthetic rat ANP both caused profound hypotension, ANP caused both natriuresis and diuresis, while chicken heart extract did not. The present study sought to locate the region(s) of chicken heart containing the hypotensive activity, and to observe the effect on sodium and water excretion and blood pressure in rats. Acid extracts of either atrium, either ventricle, ventricular septum, skeletal muscle, and liver were identically prepared from chickens and rats. Extracts were adjusted to the same protein concentration and injected (0.15 ml/kg) into anesthetized Single Comb White Leghorn roosters. Mean arterial pressure (MAP) and the time for recovery were measured. The most potent extract from chicken hearts was from the left ventricle (-38 +/- 1 mm Hg, 149 +/- 9 sec to recover). All other extracts (including right ventricle) produced only small (10-20 mm Hg), short-lived (20-30 sec) decreases in MAP. In contrast, only rat atrial extracts evoked long-lasting hypotension (greater than 40 mm Hg, recovery time greater than 200 sec). A 30-min infusion of the most potent chicken extract (left ventricle, CLV) into rats produced a small but significant natriuresis and diuresis compared to the vehicle time control (P less than 0.05) and the hypotensive response to bolus injection was about one-third that seen in the chicken. The location of potent spasmolytic activity primarily in chicken left ventricle, the different avian renal responses to chicken heart extract and synthetic rat ANP (5), and the weak diuretic, natriuretic, and hypotensive effects of CLV extract in rats all suggest that the chicken heart substance may be different from mammalian ANP.     

Acaricidal activities of extracts of Kochia scoparia against Tetranychus urticae, Tetranychus cinnabarinus, and Tetranychus viennensis (Acari: Tetranychidae)

Extracts of an annual herbaceous plant, Kochia scoparia (L.) Schrad (Macrophomina), were bioassayed to determine their acaricidal activities against Tetranychus urticae Koch, Tetranychus cinnabarinus (Boisduval), and Tetranychus viennensis Zacher (Acari: Tetranychidae) in the laboratory. Extracts had both contact and systemic toxicity to these mites. Three solvents were tested for preparing crude extracts: petroleum ether, chloroform, and methanol. Methanol was the most effective solvent, extracting 3.11-4.53% of the acaricide. Petroleum ether was the least effective solvent, extracting 1.25-1.54%. However, extracts with chloroform resulted in the highest mite mortality (78.86%), and ultrasound-assisted extraction required the least time (10 min). Concentrated extracts were prepared using chloroform, methyl acetate, or distilled water as a solvent. Mite mortalities from the concentrated extracts by methyl acetate or distilled water were significantly lower than those by chloroform. The mean lethal concentrations (LC50) of the extracts by chloroform, methyl acetate, and distilled water to the mites were 0.71 +/- 0.06, 2.08 +/- 0.16 and 8.75 +/- 0.062 mg/ml, respectively. After liquid chromatography and thin layer chromatography, the concentrated extracts by chloroform were separated into seven groups of isolated fractions and tested for acaricidal activity.     

Excretion of catecholamines in rats,mice and chicken

Stress assessment favours methods, which do not interfere with an animal's endocrine status. To develop such non-invasive methods, detailed knowledge about the excretion of hormone metabolites in the faeces and urine is necessary. Our study was therefore designed to generate basic information about catecholamine excretion in rats, mice and chickens. After administration of (3)H-epinephrine or (3)H-norepinephrine to male and female rats, mice and chickens, all voided excreta were collected for 4 weeks, 3 weeks or for 10 days, respectively. Peak concentrations of radioactivity appeared in one of the first urinary samples of mice and rats and in the first droppings in chickens 0.2-7.2 h after injection. In rats, between 77.3 and 95.6% of the recovered catecholamine metabolites were found in the urine, while in mice, a mean of 76.3% were excreted in the urine. Peak concentrations in the faeces were found 7.4 h post injection in mice, and after about 16.4 h in rats (means). Our study provides valuable data about the route and the profile of catecholamine excretion in three frequently used species of laboratory animals. This represents the first step in the development of a reliable, non-invasive quantification of epinephrine and norepinephrine to monitor sympatho-adrenomedullary activity, although promising results for the development of a non-invasive method were found only for the chicken.     

The use of the [13C]/[12C] ratio for the assay of the microbial oxidation of hydrocarbons

The study deals with a comparative analysis of the relative abundances of the carbon isotopes 12C and 13C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope composition of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n-hexadecane (delta 13C = -44.6 +/- 0.2@1000) were characterized by the isotope effects delta 13CCO2 = -50.2 +/- 0.4@1000, delta 13Cbiom = -46.6 +/- 0.4@1000 and delta 13Cexo = -41.5 +/- 0.4@1000, respectively. The isotope composition of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (delta 13C = -21 +/- 0.4@1000) were characterized by the isotope effects delta 13CCO2 = -24.1 +/- 0.4@1000, delta 13Cbiom = -19.2 +/- 0.4@1000 and delta 13Cexo = -19.1 +/- 0.4@1000, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the enviroment is discussed.     

ATP and ADP hydrolysis in fish, chicken and rat synaptosomes

Ecto-enzymes capable of hydrolyzing ATP and ADP (NTPDase) are present in the central nervous system of various species. In the present investigation we studied the synaptosomal NTPDase (ATP diphosphohydrolase, apyrase, E.C. 3.6.1.5) from fish, chicken and rats under different conditions and in the presence of several classical inhibitors. The cation concentration required for maximal activity was 0.5 mM for fish, 1.0 mM for chickens and 1.5 mM for rats with both substrates. The results showed that the pH optimum for all animal preparations was close to 8.0. The temperature used was 25–27°C for fish and 35–37°C for chicken and rat preparations. The inhibitors azide and fluoride only inhibited the preparation at high concentrations (10 mM). Lanthanum (0.1–0.4 mM), N-ethylmaleimide (0.4–3.0 mM) and ouabain (0.5–3.0 mM) had no effect on NTPDase activity from fish, chickens or rats. Orthovanadate (0.1–0.3 mM) only inhibited fish synaptosomal NTPDase. Trifluoperazine (0.05–0.2 mM) and suramin (0.03–0.3 mM) inhibited NTPDase at all concentrations tested. Suramin was the most potent compound in causing inhibition, presenting inhibition at 30 μM. Our results demonstrate that the synaptosomal NTPDase response to several factors is similar in fish, chickens and rats, and that the enzyme presents functional homology.     

Tissue-specific expression of the chicken alpha A-crystallin gene in cultured lens epithelia and transgenic mice

The present experiments show that the single gene for the lens-specific protein alpha A-crystallin of chickens and mice uses a different subset of cis- and trans-acting regulatory elements for expression in transfected embryonic chicken lens epithelial cells. A chicken alpha A-crystallin-chloramphenicol acetyltransferase (CAT) fusion gene required 162 base pairs whereas the murine alpha A-crystallin-CAT fusion gene required only 111 base pairs of 5'-flanking sequences for efficient tissue-specific expression in the transfected chicken lens cells. Gel retardation and competition experiments were performed using embryonic chicken lens nuclear extract and oligodeoxynucleotides identical to the 5'-flanking region of the chicken (-170/-111) and murine (-111/-88 and -88/-55) alpha A-crystallin gene. The results indicated that these homologous promoters use different nuclear factors for function. Methylation interference analysis identified a dyad of symmetry (CTGGTTCCCACCAG) at position -153 to -140 in the chicken alpha A-crystallin promoter which binds one or more lens nuclear factors. Gel mobility shift experiments using nuclear extracts of brain, reticulocytes, and muscle of embryonic chickens or HeLa cells suggested that the factor(s) binding to the chicken alpha A-crystallin gene promoter sequences are not lens specific. Despite differences in the functional and protein-binding properties of the alpha A-crystallin gene promoter of chickens and mice, expression of the chicken alpha A-crystallin-CAT fusion gene in transgenic mice was lens specific, consistent with a common underlying mechanism for expression of the alpha A-crystallin gene in chickens and mice.     

Age-dependent changes in cation transport in the chicken erythrocyte

Previous work has shown that the transport phenotype of chicken erythrocytes changes with the age of the chicken. Here, we report changes in the transport of choline and K+ in erythrocytes from chickens at different developmental ages. The transport of choline in chicken erythrocytes was predominantly via saturable transport systems, was highest in erythrocytes from 1-day-old chickens and declined with chicken age when tested at 2 weeks of age and in mature chickens. Both Km and Vmax values for choline transport in chicken erythrocytes declined with chicken age. Similarly, the total unidirectional influx of K+ was highest in erythrocytes from 1-day-old chickens and declined with chicken age, as did ouabain-sensitive K+ influxes, which can be attributed to the Na+/K+ pump. In isotonic conditions, bumetanide-sensitive K+ influxes, which can be attributed to the Na+-K+-2Cl- cotransporter, were only measurable in erythrocytes from 1-day-old chickens. However, when stimulated by hypertonic conditions, bumetanide-sensitive K+ influxes were essentially identical in erythrocytes from 1-day- and 2-week-old chickens but decreased in erythrocytes from mature chickens. We conclude that both choline and K+ influxes decrease significantly in erythrocytes from chickens with increasing age. The changes are substantial but complex and may involve both regulation of existing transporters, and substitution or deletion of specific transporter isoforms.     

A comparative analysis of carbon dioxide displacement rates for euthanasia of the ferret

Though carbon dioxide asphyxiation is a common method of euthanasia for laboratory animals, species-specific guidelines have not been established for this procedure in the domestic ferret (Mustela putorius furo). The authors investigated the efficacy and stress effects of carbon dioxide euthanasia in 24 ferrets that had participated in previous experimental protocols. They euthanized ferrets by placing them in cages that were either prefilled with carbon dioxide or gradually filled at a displacement rate of 10%, 20% or 50% of the cage volume per min. Blinded observers subjectively evaluated ferret distress. Prefilling the cage or filling it at a rate of 50% volume per min resulted in less time to recumbency and to last breath than did filling the cage at a slower displacement rate. Slower carbon dioxide displacement rates also caused an increase in ferret blood glucose concentrations, which may indicate distress. Overall, observers found that prefilling the euthanasia cage caused the least stress to ferrets.     

Effect of potassium deficiency on carbon dioxide,cation, and phosphate content of muscle,with a note on the carbon dioxide content of human muscle

Albino rats weighing 160 to 175 gm. were fed a complete synthetic diet containing 0.003 per cent potassium and 0.7 per cent sodium for 40 days. Controls were given the same diet plus adequate added potassium. 1. Data from analyses of serum and skeletal muscle showed (a) a fall in serum chloride concentration and an increase in serum carbon dioxide concentration and pH in the potassium-deficient rats; (b) increases of sodium, magnesium, and calcium and a decrease of potassium in the muscle of the potassium-deficient rats; (c) no change of muscle chloride or carbon dioxide concentrations in the potassium-deficient rats. (2) Application of the Wallace-Hastings calculations to these data revealed (a) intracellular pH of the skeletal muscle of the normal rat to be 6.98 +/- 0.08; (b) an increase in serum partial pressure of carbon dioxide (pCO(2)) in potassium deficiency, together with increases in concentrations of [H(2)CO(2)] and [HCO(3) (-)] per kg. extracellular water and [H(2)CO(3)] per kg. cell water; (c) a decrease in values for [CO(2)] and [HCO(3) (-)] per kg. intracellular water; (d) a fall of intracellular pH in potassium deficiency to 6.42 +/- 0.05. (3) Analyses of sacrospinalis muscle from five men undergoing operation for ruptured intervertebral disc showed a mean value of 9.46 +/- 1.31 mM carbon dioxide per kg. blood-free tissue. Some problems of interpretation of data are briefly discussed.     

Ultradian and circadian compartmentalization of respiratory and metabolic exchanges in small laboratory vertebrates

Carbon dioxide emission (VCO2) taken as an index of respiratory and metabolic exchanges, was continuously recorded during 4-30 consecutive days in 100 quail, 87 chicks, 347 rats, 665 mice and 70 guinea-pigs which were under controlled environmental parameters. Harmonic analysis, fast Fourier transform, chi-square periodograms, peak and trough intervals were computed with VCO2 values obtained with CO2 concentrations sampled every 20 min on the CO2 recordings. In LD 12:12 alternation, circadian rhythms were observed in all quail, chicks, rats and mice, but only in 80% of the guinea-pigs. Ultradian VCO2 rhythms, with periods which show statistically significant interspecies differences, were assessed. For each of the 5 species these computed periods, which were the same in LL and DD, were: 1.17 h for quail and chickens, 1.25 h for rats, 1.50 h for mice and 1.0 h for guinea-pigs. In LD 12:12 these periods were different during L and D in quail, chicks, rats and mice, but not in guinea-pigs. The amplitudes of these ultradian variations were, according to the species, 10-20% of their mean VCO2 levels. These ultradian rhythms persist in the absence (or masking) of circadian rhythms, e.g. in LD 12:12 in 20% of guinea-pigs and in LL in 87% of Japanese quail and in 23% of Sprague-Dawley rats. Moreover, these ultradian rhythms persist during starvation, locomotor activity restraint and ageing. These ultradian VCO2 cycles which are related to rest-activity variations appear to be basic physiological rhythms with a genetic origin.     

The effect of euthanasia technique on vascular arachidonic acid metabolism and vascular and intestinal smooth muscle contractility

This study was designed to determine the effects that specific euthanasia methods have on vascular arachidonic acid metabolism and vascular and intestinal smooth muscle contractility. Rats were euthanatized by decapitation (DC), pentobarbital overdose (PB), or anesthesia with CO2, methoxyflurane or ether followed by DC (CO2-DC, Met-DC, Ether-DC, respectively). Rabbits were killed by a similar protocol, but CO2 overexposure replaced Ether-DC. The rat and rabbit aortas produced mainly 6-keto PGF1 alpha, the prostacyclin metabolite, and lesser amounts of PGE2. No qualitative differences were seen in arachidonate metabolites. However, aortic tissue from rabbits and rats killed by Met-DC produced more prostacyclin. In contrast, aorta from rabbits euthanatized by CO2-DC produced less prostacyclin than controls, whereas aorta from rats killed in the same way yielded greater amounts of prostacyclin. Aortic tissue from rabbits killed by Met-DC and CO2-OD was less responsive to acetylcholine (ACH). Intestinal contractility to ACH was increased in rabbits when Met-DC was used as the method of euthanasia, while colon from rats sacrificed by Met-DC showed decreased responsiveness to ACH. Colon from rats killed by intraperitoneal PB exhibited altered contractility to ACH and norepinephrine. The results of this study show that methoxyflurane, carbon dioxide (rabbit) and pentobarbital (rat) alter the vascular synthesis of prostacyclin and smooth muscle contractility. We conclude that the method of euthanasia affects certain physiologic parameters and careful consideration should be given to the selection of a particular euthanasia technique.     

Myocardial contractility in chickens (Gallus gallus): analysis of systolic time intervals

The avian cardiovascular system is of special interest because avian hearts are relatively larger than mammalian hearts, and activation of ventricular myocardium in birds has a "flash" pattern. Systolic time intervals and indices of myocardial contractility were examined in anaesthetized open-chest chickens by polycardiography, including synchronous recordings of electrocardiogram, phonocardiogram, and apex cardiogram. The asynchronous contraction time, isometric contraction time, pre-ejection period and ejection time were 26 +/- 3 (Mean +/- SD), 21 +/- 9, 47 +/- 12, and 83 +/- 23 ms, respectively, for heart rates of 260 +/- 57 bpm. The myocardial tension index, isometric contraction index and the pre-ejection period/ejection time ratio were 0.39 +/- 0.11, 0.42 +/- 0.10, and 0.54 +/- 0.14, respectively. A "flash" pattern of ventricular myocardial depolarization causes more rapid excitation and as a consequence shorter asynchronous contraction time of relatively larger chicken hearts compared with rabbit hearts. Inverse relation (P < 0.05) of the asynchronous contraction time to the heart rate in chickens is probably associated with the specific activation pattern of avian ventricles. Establishment of the values of systolic time intervals will facilitate a better understanding of cardiac function in birds. The obtained results are discussed in comparison with the rabbit. The indices calculated from the systolic time intervals show disadvantageous contractile function of chicken heart compared to rabbit heart.     

The response of the blood-sucking bug Triatoma infestans to carbon dioxide and other host odours

Behavioural responses of Triatoma infestans larvae to carbon dioxide and other odours of vertebrate origin were investigated in a locomotion compensator. T. infestans oriented towards airstreams enriched with carbon dioxide exhibiting a threshold response between 300 and 400 p.p.m. above the ambient CO(2) background. The accuracy of the oriented response to carbon dioxide improved with stimulus intensity. Remarkably, insects did not show any change in their sensitivity threshold to carbon dioxide with the starvation time. The attractiveness to carbon dioxide depended on the time of the day, i.e. these nocturnal bugs only oriented towards carbon dioxide-loaded airstreams during the first hours of the scotophase. L-lactic acid did not evoke oriented responses when it was presented as a single stimulus in a wide range of intensities. However, a marked synergism was evident when L-lactic acid was combined with a sub-threshold concentration of carbon dioxide. Under this condition, the threshold response to carbon dioxide decreased to 75-150 p.p.m. above ambient CO(2) background. The isomer D-lactic acid evoked no response, either alone or in combination with carbon dioxide. When insects were stimulated with 1-octen-3-ol a significant positive orientation was found. This response was not modified by the addition of carbon dioxide.     

Euthanasia of rats with carbon dioxide--animal welfare aspects

A method of inducing euthanasia by carbon dioxide (CO2) inhalation in the home cage of an animal is described and tested for distress by behavioural as well as by hormonal measures. The animals were maintained in their home cage while CO2 was induced at a flow of 6 l/min. The behaviour of the animals was measured continuously as were the serum concentrations of glucose, ACTH and corticosterone 30, 75 and 120 s after the CO2 was introduced into the cage. In order to test for distress, two groups of rats were pre-treated with acepromazine (orally) and pentobarbiturate (i.p. injection) respectively, in order to reduce possible distress caused by CO2 euthanasia, and were compared with control groups. There were no signs of distress by behavioural or by hormonal changes. All changes seen could be attributed to experimental effects and, especially as there was no difference between the pre-treated and the control groups of rats, it must be assumed that the described method of euthanasia is in concordance with animal welfare, it leads to rapid death without severe distress or pain, and it seems therefore to be 'humane'.     

Seroprevalence and isolation of Toxoplasma gondii from free-range chickens in Ghana, Indonesia, Italy, Poland, and Vietnam

The prevalence of Toxoplasma gondii in free-ranging chickens (Gallus domesticus) is a good indicator of the prevalence of the parasite's oocysts in soil because chicken feed from the ground. The prevalence of T. gondii in free-range chickens from Ghana, Indonesia, Italy, Poland, and Vietnam was determined using the modified agglutination test (MAT). Antibodies to T. gondii were found in 41 (64%) of 64 chickens from Ghana, 24 (24.4%) of 98 chickens from Indonesia, 10 (12.5%) of 80 chickens from Italy, 6 (30%) of 20 chickens from Poland, and 81 (24.2%) of 330 chickens from Vietnam. Hearts and brains of chickens were bioassayed for T. gondii. Viable T. gondii was isolated from 2 chickens from Ghana, 1 chicken from Indonesia, 3 chickens from Italy, 2 chickens from Poland, and 1 chicken from Vietnam. Toxoplasma gondii isolates from 9 chickens were genotyped using 10 PCR-RFLP markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. A total of 7 genotypes was identified; the 3 isolates from chickens from Italy were clonal type II, and the others were nonclonal. This is the first report of genetic characterization of T. gondii isolates from animals from these countries.     

Efficiency of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines

The antagonistic effect exerted towards Salmonella typhimurium by the flora issued from conventional chickens was studied in gnotobiotic animals. In germfree chickens and mice inoculated with S. typhimurium, the highest bacterial counts were observed in ceca, and were not significantly different in either host. The protection afforded by the inoculation of cecal flora issued from a conventional chicken was more effective when this flora was inoculated first into germfree chickens than when it was given only after inoculation with S. typhimurium. Administration of a cecal flora from a 15-day-old chick to gnotobiotic mice and chicken resulted in the inhibition of a further intestinal colonization by S. typhimurium in both hosts. Sixteen strains were isolated among the predominant populations of the fecal flora from chicken flora recipient mice. Association of 14 strains of strictly anaerobic bacteria with 2 strains of Escherichia coli and Streptococcus faecium only decreased the number of S. typhimurium in the ileum of gnotobiotic mice, but not in their cecum. Anaerobe cultures were obtained from 10(-6) and 10(-8) dilutions prepared from the fecal flora of gnotobiotic recipient mice. Antagonistic bacteria were present only in cultures from the 10(-6) dilution. Cecal concentrations of volatile fatty acids were shown not to be the sole factor implicated in the antagonistic effect against S. typhimurium.     

Acaricidal activity of Juglans regia leaf extracts on Tetranychus viennensis and Tetranychus cinnabarinus (Acari: Tetranychidae)

Leaf extracts of the walnut, Juglans regia L., were evaluated under laboratory conditions to determine their acaricidal activity on the mites Tetranychus cinnabarinus (Boisduval) and Tetranychus viennensis Zacher (Acari: Tetranychidae). Extracts had both contact and systemic toxicity to these mites. The four solvents tested for preparing crude extracts were petroleum ether, chloroform, ethyl acetate, and methanol. Methanol was the most efficient solvent, with an extraction rate from 17.06 + 0.80 to 20.27 +/- 0.28%. Petroleum ether was the least effective solvent, with extraction rates from 2.30 +/- 0.13 to 2.71 +/- 0.13%. However, the crude extracts with petroleum ether resulted in the highest mite mortality (79.04 +/- 0.52%) in a slide dip bioassay. Mites mortalities from the concentrated extracts prepared by chloroform, ethyl acetate, methanol, or distilled water were significantly lower than petroleum ether. The mean lethal concentrations (LC50) of the extracts from petroleum ether, chloroform, ethyl acetate, methanol, and distilled water to the two mite species were 0.73 +/- 0.04, 1.66 +/- 0.28, 4.96 +/- 0.35, 7.45 +/- 0.67, and 9.91 +/- 0.32 mg/ml, respectively. After liquid chromatography and thin-layer chromatography, the concentrated extracts of petroleum ether were separated into eight fractions and tested for acaricidal activity. Fraction 6 produced significantly higher mite mortality rates than the other groups, killing approximately 90% of both species.     

Combining Nitrous Oxide with Carbon Dioxide Decreases the Time to Loss of Consciousness during Euthanasia in Mice — Refinement of Animal Welfare?

Carbon dioxide (CO(2)) is the most commonly used euthanasia agent for rodents despite potentially causing pain and distress. Nitrous oxide is used in man to speed induction of anaesthesia with volatile anaesthetics, via a mechanism referred to as the "second gas" effect. We therefore evaluated the addition of Nitrous Oxide (N(2)O) to a rising CO(2) concentration could be used as a welfare refinement of the euthanasia process in mice, by shortening the duration of conscious exposure to CO2. Firstly, to assess the effect of N(2)O on the induction of anaesthesia in mice, 12 female C57Bl/6 mice were anaesthetized in a crossover protocol with the following combinations: Isoflurane (5%)+O(2) (95%); Isoflurane (5%)+N(2)O (75%)+O(2) (25%) and N(2)O (75%)+O(2) (25%) with a total flow rate of 3 l/min (into a 7 l induction chamber). The addition of N(2)O to isoflurane reduced the time to loss of the righting reflex by 17.6%. Secondly, 18 C57Bl/6 and 18 CD1 mice were individually euthanized by gradually filling the induction chamber with either: CO(2) (20% of the chamber volume.min-1); CO(2)+N(2)O (20 and 60% of the chamber volume.min(-1) respectively); or CO(2)+Nitrogen (N(2)) (20 and 60% of the chamber volume.min-1). Arterial partial pressure (P(a)) of O(2) and CO(2) were measured as well as blood pH and lactate. When compared to the gradually rising CO(2) euthanasia, addition of a high concentration of N(2)O to CO(2) lowered the time to loss of righting reflex by 10.3% (P<0.001), lead to a lower P(a)O(2) (12.55 ± 3.67 mmHg, P<0.001), a higher lactataemia (4.64 ± 1.04 mmol.l(-1), P = 0.026), without any behaviour indicative of distress. Nitrous oxide reduces the time of conscious exposure to gradually rising CO(2) during euthanasia and hence may reduce the duration of any stress or distress to which mice are exposed during euthanasia.