A comparison of rodent caging systems based on microenvironmental parameters.

Four different mouse caging systems were evaluated for microenvironmental temperature, carbon dioxide, relative humidity (RH) and ammonia levels during a 7-day testing period. All caging systems evaluated had polycarbonate bases and consisted of either a molded polyester (MP) filter lid, one of two different polycarbonate filter lids, or no filter lid which served as a control. At 50% macroenvironmental RH (study I), all systems maintained an intracage temperature of 75.5 degrees F +/- 0.5 degrees. Both polycarbonate systems averaged greater than 2200 ppm of carbon dioxide more than the MP system and the controls. When compared with RH in the control cages, RH levels averaged over 20% and 5 to 8% RH greater in the polycarbonate filter lid systems and the MP system, respectively. There were no appreciable ammonia levels in either the MP or control systems. In the polycarbonate filter lid systems, ammonia levels were detectable on day 4 and were greater than 200 ppm by day 6. At 20% macroenvironmental RH (study II), there was a proportional 15 to 30% RH decrease from study I levels. Ammonia levels were undetectable in any system until day 7 and averaged only 17 ppm in one of the polycarbonate systems. Minimal differences were observed in studies III, IV and V when pine shavings were used instead of hardwood chips, a CD-1 stock instead of a DBA/2J strain, and different grades of filter inserts in the polycarbonate systems, respectively.     

The impact of reduced frequency of cage changes on the health of mice housed in ventilated cages

Our purpose in this investigation was to determine if we could reduce cage changing frequency without adversely affecting the health of mice. We housed mice at three different cage changing frequencies: 7, 14, and 21 days, each at three different cage ventilation rates: 30, 60 and 100 air changes per hour (ACH), for a total of nine experimental conditions. For each condition, we evaluated the health of 12 breeding pairs and 12 breeding trios of C57BL/6J mice for 7 months. Health was assessed by breeding performance, weanling weight and growth, plasma corticosterone levels, immune function, and histological examination of selected organs. Over a period of 4 months, we monitored the cage microenvironment for ammonia and carbon dioxide concentrations, relative humidity, and temperature one day prior to changing the cage. The relative humidity, carbon dioxide concentrations, and temperature of the cages at all conditions were within acceptable levels. Ammonia concentrations remained below 25 ppm (parts per million) in most cages, but, even at higher concentrations, did not adversely affect the health of mice. Frequency of cage changing had only one significant effect; pup mortality with pair matings was greater at the cage changing frequency of 7 days compared with 14 or 21 days. In addition, pup mortality with pair matings was higher at 30 ACH compared with other ventilation rates. In conclusion, under the conditions of this study, cage changes once every 14 days and ventilation rates of 60 ACH provide optimum conditions for animal health and practical husbandry.     

Effects of an individually ventilated cage system on the airway integrity of rats (Rattus norvegicus) in a laboratory in Brazil

The ventilation method used in the management of laboratory rats is important in maintaining their health. Rats kept under general diluting ventilation (GDV) are exposed to high levels of pollutants present in the environment (dust, airborne bacteria, etc.) or those pollutants produced by animal metabolism and excretion inside the boxes (e.g. ammonia and carbon dioxide). These pollutants may contribute to respiratory pathologies. An alternative experimental ventilation system for laboratory animal housing using intracage ventilation technology (individually ventilated cage system, IVC) was developed. In this system, ammonia levels decreased and rats exhibited better reproductive performance and a lower incidence of pneumonia than rats maintained under GDV. Using two different levels of air speed (0.03-0.26 m/s: IVC(1); 0.27-0.80 m/s: IVC(2)), the effects of IVC were compared with GDV (control) in Wistar rats in terms of respiratory mucus properties, on the nasal epithelium (as measured by quantitative morphometry) and on the lungs (as determined by the cellular composition obtained by bronchoalveolar lavage). Mucus of the respiratory system was evaluated using the following techniques: rheology (viscoelasticity) by microrheometer, in vitro mucociliary transportability (frog palate) and contact angle (an indicator of adhesivity). Also, membrane transepithelial potential difference was measured as a biomarker of airway integrity. After bedding was changed, ammonia concentrations inside the cages on day 3 were significantly higher for GDV than for IVC(1) and IVC(2). The potential-difference values for IVC(1), IVC(2) and GDV in the epiglottis and in the trachea also showed differences. Although some significant differences were observed across the three groups in counts of some cell types, the intragroup results were highly variable among individuals and inconsistent between sexes. No significant differences in the other parameters were found across groups. These results establish that rats maintained under GDV in relatively unregulated conditions are exposed to factors that can lead to deleterious effects on the ciliated epithelium of the airways, and that these effects can be prevented by the use of IVC.     

Microclimate in two types of rat cages

The microclimate in two types of rat cages (a Makrolon type IV with a solid floor and a stainless steel cage with a wire mesh floor (five rats per cage)) placed in the same macro-environment was compared. The temperature, relative humidity and ammonia concentration in the cages were measured twice a day for 8 days. The cages were cleaned every 4 days. The greatest difference between the cage types was in the ammonia build-up. In Makrolon cages the ammonia concentration never reached 5 ppm, whereas in steel cages it showed a constant increase and already exceeded the threshold limit for man (25 ppm for 8 h per day) on the third day after cleaning.     

Cage ammonia levels during murine reproduction.

Monogamous pairs of inbred BALB/c and outbred CD-1 mice housed in M2 cages with a floor area of 330 cm2, produced a mean cage ammonia level of 26 ppm and 154 ppm respectively over a four day period prior to weaning their litters. A gradient of ammonia exists from the nest to the food hopper. By housing CD-1 monogamous pairs in RM2 cages which have double the floor area of M2 cages (676 cm2), a lower mean level of ammonia was recorded at the same stage of reproduction and air sampling. The CD-1 mice in particular, were subjected to high levels of ammonia when compared with long term human health and safety occupational exposure limits of 25 ppm.     

A forced-air individually ventilated caging system for rodents

The practice of using a protective filter apparatus when housing five mice per cage resulted in ammonia levels exceeding 25 ppm. A forced-air individually ventilated caging system was constructed using polyvinyl-chloride tubing fitted to a rodent rack. The ammonia level was decreased using this ventilation system.     

The effects of different rack systems on the breeding performance of DBA/2 mice

Housing systems for laboratory animals have been developed over a long time. Micro-environmental systems such as positive, individually ventilated caging systems and forced-air-ventilated systems are increasingly used by many researchers to reduce cross contamination between cages. There have been many investigations of the impact of these systems on the health of animals, the light intensity, the relative humidity and temperature of cages, the concentration of ammonia and CO(2), and other factors in the cages. The aim of the present study was to compare the effects of different rack systems and to understand the influence of environmental enrichment on the breeding performance of mice. Sixty DBA/2 breeding pairs were used for this experiment. Animals were kept in three rack systems: a ventilated cabinet, a normal open rack and an individually ventilated cage rack (IVC rack) with enriched or non-enriched type II elongated Makrolon cages. Reproduction performance was recorded from 10 to 40 weeks of age. In all three rack systems there was a similar breeding index (pups/dam/week) in non-enriched groups during the long-term breeding period, but the coefficients of variation in the IVC rack were higher for most parameters. This type of enrichment seems to lead to a decrease in the number of pups born, especially in the IVC group. However, there was no significant difference in breeding index (young weaned/female/week).     

Ammonia as an attractive component of host odour for the yellow fever mosquito, Aedes aegypti

Behavioural responses of Aedes aegypti mosquitoes to ammonia were investigated in a modified Y-tube olfactometer. Ammonia was attractive in concentrations from 17 ppb to 17 ppm in air when presented together with lactic acid. Aqueous solutions of ammonia salts in concentrations comparable to those found in human sweat also increased the attractiveness of lactic acid. The role of lactic acid as an essential synergist for ammonia became further apparent by the fact that ammonia alone or in combination with carbon dioxide was not effective, even though the synergistic effect of carbon dioxide and lactic acid was corroborated. An extract from human skin residues, which attracts approximately 80% of the tested mosquitoes, contains both lactic acid and ammonia. The combination of these compounds, however, attracts no more than 45%, indicating that other components on human skin also play a role in host finding. Preparative liquid chromatography of the skin extract yielded three behaviourally active fractions which work together synergistically. Fraction III contains lactic acid as the effective principle; the compositions of the other two have not been clarified yet. The attractiveness of fraction I was augmented considerably when ammonia was added, whereas the effect of fraction II was not influenced by ammonia. These results suggests that ammonia is part of the effective principle of fraction II and contributes to the attractive effect of host odours.     

The effects of different types of individually ventilated caging systems on growing male mice

Ventilation rate and turnover rate of dry air vary among different types of ventilation systems used with individually ventilated cages (IVCs) and can affect the well-being of rodents housed in these cages. The authors compared the effects of two types of IVC systems, forced-air IVCs and motor-free IVCs, on 4-week-old C57Bl/6J male mice. The mice were acclimatized to the cages for 8 d and then monitored for 87 d. Their body weights, food and water consumption and preferred resting areas were recorded. Mice that were housed in motor-free IVCs had a significantly greater increase in body weight than those housed in forced-air IVCs, despite having similar food consumption. Mice in forced-air IVCs had greater water consumption than mice in motor-free IVCs. In addition, mice in forced-air IVCs were more frequently located in the front halves of their cages, whereas mice in motor-free IVCs were located with similar frequency in the front and back halves of their cages, perhaps because of the higher ventilation rate or the location of the air inlets and outlets in the rear of the cage. These results suggest that body weight, food and water consumption and intracage location of growing male mice are influenced by the type of ventilation system used in the cages in which the mice are housed.     

Animal chamber for in vivo testing of toxic gases

A closed-circuit chamber system for exposing animals to toxic concentrations of oxygen or nitrogen was designed and built. The facility was equipped with systems for the removal of carbon dioxide, ammonia, methane, water vapor, and particulates from the gas stream. Integral systems were built to monitor and control temperature (+/- 1 degree C), relative humidity (+/- 2%), oxygen concentration (10 to 100% +/- 0.5%), and methane concentration (below 2%). Operation was set at standard pressure (1.0 X 10(5) Newtons/m2). Hyperoxic studies have been performed on six species of rodents, as well as sheep, and have lasted from 1 to 28 days.     

Methylamine metabolism in Hansenula polymorpha: an in vivo 13C and 31P nuclear magnetic resonance study.

Methylamine uptake, oxidation, and assimilation were studied in Hansenula polymorpha, a methylotrophic yeast. The constitutive ammonia transport system was shown to be effective at accumulating methylamine within cells cultured with methylamine or ammonia as a nitrogen source. [13C]methylamine oxidation rates were measured in vivo in methylamine-adapted cells by 13C nuclear magnetic resonance and were found to be lower than its uptake rate into the cells. The 13C label of methylamine was found exclusively in trehalose and glycerol, and [13C]formaldehyde was also extensively assimilated, indicating the presence of an assimilation pathway for the methylamine carbon. In vivo 31P nuclear magnetic resonance analysis showed major differences in the endogenous polyphosphate levels and mean chain length during adaptation of the cells from ammonia to methylamine, indicating that methylamine accumulated in the vacuole in the same manner as basic amino acids and purines. [13C]glucose metabolism was drastically altered during adaptation of the cells from ammonia to methylamine as a nitrogen source. The total rate of glucose utilization and the rate of ethanol production fell. Direct trehalose synthesis from glucose increased, indicating a switch from carbon utilization for growth to that for storage. The rate of methylamine oxidation was sufficient to support a much higher flow of carbon into central biosynthetic pathways. These results suggest that this reduction in biosynthetic carbon flow, rather than nitrogen availability, was the main factor responsible for reducing the growth rate of the yeast when ammonia was replaced by methylamine as the nitrogen source.     

Effects of housing density on nasal pathology of breeding mice housed in individually ventilated cages

The 2011 edition of the Guide for the Care and Use of Laboratory Animals includes new recommendations for the amount of floor space that should be provided to breeding mice. When pairs or trios of continuously breeding mice are housed in shoebox cages, they may have less than this recommended amount of floor space. High housing densities may adversely affect animal health, for example, by compromising air quality inside the cage. Hence, some institutions are carefully reevaluating the microenvironments of breeding cages. The use of individually ventilated cages (IVCs) to house research mice allows for greater control over the quality of the cage microenvironment. The authors evaluated the microenvironments of shoebox cages in an IVC rack system housing breeding and non-breeding Swiss Webster mice. Ammonia concentrations were significantly higher in cages housing breeding trios with two litters. Histopathologic lesions attributable to inhaled irritants such as ammonia were found in mice housed in breeding pairs and trios. The authors conclude that the microenvironments of cages in an IVC rack system housing breeding pairs and trios may be detrimental to animal health.     

Influence of Pathological Concentrations of Ammonia on Metabolic Fate of 14C-Labeled Glutamate in Astrocytes in Primary Cultures

Rates of glutamine formation and of carbon dioxide production (as an indication of oxidative deamination of glutamate) were determined in primary cultures of astrocytes exposed to 50 microM labeled glutamate in the absence or presence of added ammonia (0.1-3 mM). Glutamine formation (1.7 nmol/min/mg protein) was unaffected by all concentrations of added ammonia. This probably reflects the presence of a low content of ammonia (0.1-0.2 mM), originating from degradation of glutamine, in the cells even in the absence of added ammonia, and it shows that pathophysiological concentrations of ammonia do not increase the formation of glutamine from exogenous glutamate. The carbon dioxide production rate was 5.9 nmol/min/mg protein, i.e., three to four times higher than the rate of glutamine formation. It was significantly reduced (to 3.5 nmol/min/mg protein) in the presence of 1 mM or more of ammonia. This is in keeping with suggestions by others that toxic levels of ammonia affect oxidative metabolism.     

The growth of pestalotia rhododendri Guba in relation to volatile metabolites

Summary A strain of Pestalotia rhododendri Guba produced good growth in a simple glucose-mineral-salt medium with the addition of thiamine.The fungus was grown on agar medium in Ryan-tubes and through some of the tubes an airstream was passed. For optimal growth an airflow rate of at least 60 ml/min was necessary.The growth-promoting effect of aeration was studied in relation to pH of the medium, relative humidity, oxygen supply, carbon dioxide accumulation and various nitrogen sources.No volatile, organic metabolite stimulating the growth was found.On a medium with a low carbon and a high nitrogen content, aeration made growth of Pestalotia possible when nonaerated mycelia were nearly completely inhibited. In an unbuffered medium accumulation of ammonia could explain the inhibition of growth.The agar or impurities in the agar contributed to the nitrogen nutrition of the fungus.     

Estimation of Aeration and Agitation Conditions in Respect to Oxygen Supply and Ventilation

In inosine fermentation, the yield of the product was closely related to the partial pressure of carbon dioxide in the culture system rather than the bicarbonate ion concentration in the culture liquid. The inhibitory effect of carbon dioxide was restored by the methods of reducing the partial pressure of carbon dioxide lower than a certain level. Both ventilation and chemical absorption were applicable for the restoration of the carbon dioxide inhibition, but ventilation had great advantages over the other method from an industrial view-point. In the estimation of aeration-agitation conditions for the scale-up of submerged fermentation in which carbon dioxide was inhibitory, the rate of aeration was to be established to make sufficient ventilation and overcome this inhibition. A scheme for the procedure for the estimation of aeration-agitation conditions under the consideration of influences of carbon dioxide on submerged fermentation was proposed in this paper.     

Semiparametric Modeling of Daily Ammonia Levels in Naturally Ventilated Caged-Egg Facilities

Ammonia concentration (AMC) in poultry facilities varies depending on different environmental conditions and management; however, this is a relatively unexplored subject in Colombia (South America). The objective of this study was to model daily AMC variations in a naturally ventilated caged-egg facility using generalized additive models. Four sensor nodes were used to record AMC, temperature, relative humidity and wind speed on a daily basis, with 10 minute intervals for 12 weeks. The following variables were included in the model: Heat index, Wind, Hour, Location, Height of the sensor to the ground level, and Period of manure accumulation. All effects included in the model were highly significant (p<0.001). The AMC was higher during the night and early morning when the wind was not blowing (0.0 m/s) and the heat index was extreme. The average and maximum AMC were 5.94±3.83 and 31.70 ppm, respectively. Temperatures above 25°C and humidity greater than 80% increased AMC levels. In naturally ventilated caged-egg facilities the daily variations observed in AMC primarily depend on cyclic variations of the environmental conditions and are also affected by litter handling (i.e., removal of the bedding material).     

Regulation of Senescence in Carnation (Dianthus caryophyllus): Effect of Abscisic Acid and Carbon Dioxide on Ethylene Production

Abscisic acid hastened senescence of carnation flowers and this was preceded by stimulation of accelerated ethylene production. Carbon dioxide delayed the onset of autocatalytic ethylene production in flowers regardless of treatment with abscisic acid. Flowers exhibited a low and transient climacteric of ethylene production without wilting while in 4% carbon dioxide and underwent accelerated ethylene production culminating in wilting when removed from carbon dioxide. Hypobaric ventilation, which lowers ethylene to hyponormal levels within tissues, extended flower longevity and largely negated enhancement of senescence by abscisic acid. Supplementing hypobarically ventilated flowers with ethylene hastened senescence irrespective of abscisic acid treatment. Collectively, the data indicate that abscisic acid hastens senescence of carnations largely as a result of advancing the onset of autocatalytic ethylene production.     

Gas phase composition effects on suspension cultures of Taxus cuspidata

The effect of different concentrations and combinations of oxygen, carbon dioxide, and ethylene on cell growth and taxol production in suspension cultures of Taxus cuspidata was investigated using several factorial design experiments. Low head space oxygen concentration (10% v/v) promoted early production oftaxol. High carbon dioxide concentration (10% v/v) inhibited taxol production. The most effective gas mixture composition in terms of taxol production was 10% (v/v) oxygen, 0.5% (v/v) carbon dioxide, and 5 ppm ethylene. Cultures grown underambient concentration of oxygen had a delayed uptake of glucose and fructose compared to cultures grown under 10% (v/v) oxygen. Average calcium uptake rates into the cultured cells decreased and average phosphate uptake rates increased as ethylene was increased from 0 to 10 ppm. These results may indicate that gas composition alters partitioning of nutrients, which in turn affects secondary metabolite production. (c) 1995 John Wiley & Sons, Inc.     

Impact of nitrogen addition on growth and carbon sequestration potential of Syzygium cumini under different carbon dioxide environment conditions

Human activities have significantly accelerated emissions of each carbon dioxide and biologically reactive nitrogen into the atmosphere. As nitrogen availability frequently limits forest productivity, it has long been anticipated that anthropogenic nitrogen deposition ought to stimulate carbon sequestration in forests. The present study aimed to understand the effects of nitrogen addition on the growth of the Syzygium cumini in ambient (408 ppm) and elevated (450 ppm, 500 ppm, 550 ppm and 600 ppm) carbon dioxide conditions. The addition of nitrogen in the form of potassium nitrate in different levels (0, 3, 6, 12, & 24 g N m^?2) was used in this study. The responses of the plant in terms of growth (shoot length, leaf length and leaf width) were examined. Multiple regression equation was used for the model fit to determine the carbon content. The carbon content was found to be higher in all ambient and elevated CO₂ conditions at the nitrogen addition level of 24 g N m^?2.     

Expression of arabidopsis cytosolic ascorbate peroxidase gene in response to ozone or sulfur dioxide

The effects of ozone or sulfur dioxide on antioxidant enzymes were investigated in Arabidopsis thaliana. Plants were fumigated with 0.1–0.15 ppm ozone or sulfur dioxide up to about 1 week in an environment-controlled chamber. Both pollutants increased the activities of ascorbate peroxidase and guaiacol per-oxidase in leaves, but had little effect on the activities of superoxide dismutase, catalase, monodehydroascorbate reductase, dehydroascorbate reductase or glutathione reductase. Ozone was more effective than sulfur dioxide in increasing the activities of the peroxidases. Ascorbate peroxidase activity increased 1.8-fold without a lag period during fumigation with 0.1 ppm ozone, while guaiacol peroxidase activity increased 4.4-fold with a 1-day lag. Expression of the APX1 gene encoding cytosolic ascorbate peroxidase was further investigated. Its protein levels in leaves exposed to 0.1 ppm ozone for 4 or 8 days were 1.5-fold higher than in controls. Both ozone and sulfur dioxide elevated APX1 mRNA levels in leaves at 4 and 7 days, whereas at 1 day only ozone was effective. The induction of APX1 mRNA levels by ozone (3.4- to 4.1-fold) was more prominent than that by sulfur dioxide (1.6-to 2.6-fold). The APX1 mRNA level increased by day and decreased by night. Exposure of plants to 0.1 ppm ozone enhanced the APX1 mRNA level within 3 h, which showed a diurnal rhythm similar to that of the control. These results demonstrate that near-ambient concentrations of ozone as well as similar concentrations of sulfur dioxide can induce APX1 gene expression in A. thaliana.Environmental Biology Division