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Poult Sci 2008. 87:1804-1809. doi:10.3382/ps.2007-00420
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METABOLISM AND NUTRITION: Research Note

A Method for Collecting Breath Samples from Individual Chickens for Analysis of 13CO2, H2, and CH4

R. J. Hughes1, D. R. Tivey2 and R. N. Butler3

South Australian Research and Development Institute-Pig and Poultry Production Institute Nutrition Research Laboratory, University of Adelaide, Roseworthy, SA 5371, Australia

1 Corresponding author: hughes.bob{at}saugov.sa.gov.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study describes experiments using simple helmets to collect breath samples from individual birds for measurement of 13CO2, H2, and CH4, which form the basis for various diagnostic tests for intestinal dysfunction in humans. Peak enrichment in 13C in breath CO2 occurred between 5 and 30 min postingestion by 18-d-old chickens administered a gelatin capsule containing approximately 3.6 mg of 13C-octanoic acid dissolved in vegetable oil. For 25-d-old chickens given 10 mL of homogenized cooked corn by oral gavage, peak enrichment occurred 60 to 90 min postingestion. In fully fed 25-d-old chickens, H2 and CH4 concentrations in breath ranged from 7 to 115 ppm and from 0 to 5.5 ppm, respectively. Following an overnight fast, H2 and CH4 concentrations in breath ranged from 0.5 to 7.5 ppm and 0 to 3.0 ppm, respectively, in the same chickens. Ranges in H2 (1.0 to 56.5 ppm) and CH4 (0 to 8.0 ppm) concentrations widened considerably 3 h after oral gavage with approximately 130 mg of lactulose (an indigestible disaccharide) dissolved in 5 mL of water. The results from these investigations indicate that collection of re-breathed air samples from chickens is plausible, which opens the way for development of noninvasive methods for evaluating gastrointestinal functions in chickens.

Key Words: poultry • expired breath • carbon dioxide • hydrogen • methane


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Clinicians have known for many years that unusual breath odor is often an indication of gastrointestinal dysfunction in patients (Tivey and Butler, 1999). Analysis of expired breath for nonodorous gases is now a commonly used, noninvasive diagnostic method in human medicine (Amarri and Weaver, 1995; Swart and van den Berg, 1998). Tivey and Butler (1999) concluded that breath tests should also prove to be a powerful analytical tool for nutrition research and veterinary diagnostics in domestic and agricultural species.

Breath tests involving stable isotopes are safe alternatives to radio-scintigraphy, particularly for infants and pregnant women, and when multiple or frequent tests are required (Amarri and Weaver, 1995; Swart and van den Berg, 1998). The tests involve ingestion of a substrate enriched in a stable isotope such as 13C that is relevant to the particular rate-limiting intestinal process under investigation, followed by serial breath sampling. For example, 13C-triglyceride is used to examine pancreatic lipase function (Vantrappen et al., 1989), and lactose 13C-ureide is used for measuring small intestinal transit time (Heine et al., 1995). Isotope is released as 13CO2 by a series of metabolic processes following digestion and absorption of labeled feedstuffs, and then transported via the bloodstream to the lungs for excretion. The breath samples are analyzed with an isotope-ratio mass spectrometer. The ratio of 13C and 12C isotopes in the breath is directly related to functionality of the gut in terms of release of digestive enzymes, epithelial function, or digesta transit time, all of which are measured individually by this technology (Amarri and Weaver, 1995; Swart and van den Berg, 1998).

Breath tests based on release of H2 and CH4 are also used routinely in medical practice (Robb and Davidson, 1987). For example, breath H2 measurement is used as an indicator of carbohydrate malabsorption in humans and is used for estimating the rate of passage of digesta through the small intestine. These breath tests are based on release of H2 following microbial fermentation of carbohydrates such as lactulose, which is a synthetic disaccharide not absorbed in the small intestine (Wutzke et al., 1997). Hydrogen and CH4 are not produced by mammalian or avian tissue. Studies on humans and other species indicate that samples of breath can be taken with simple, inexpensive equipment and remain stable for long periods, enabling these tests to be used in the field (Tivey and Butler, 1999). To date, there is scant information in the scientific literature on the use of breath tests on agricultural species of animals.

The aim of the present study was to devise equipment and procedures for collecting breath samples from young broiler chickens for measurement of concentrations of 13CO2, H2, and CH4, which form the basis for various diagnostic tests for intestinal dysfunctions in humans.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Development of Equipment and Procedures for Collecting Breath Samples from Chickens

The Animal Ethics Committees of the University of Adelaide and the Department of Primary Industries South Australia approved the procedures and experimental designs used in these studies. Breath testing in adult humans involves the collection of samples by asking the patient to blow through a tube into a collection vessel. When it is not possible to get a sample by voluntary means from human or animal subjects, face masks can suffice. An alternative approach is to use a nasal prong to draw breath into a syringe during exhalation. Initial attempts to construct face masks highlighted some of the practical difficulties associated with this approach to collect breath samples from chickens. It soon became clear that masks needed to fit closely to gather a sufficient quantity of breath without leakage. Furthermore, the awkward profile of the head and beak of chickens made this difficult to achieve in a mask that could be taken on and off easily without stressing the birds. The alternative of leaving the mask in place for 3 or more hours required either a pressurized air supply or a system of one-way valves to enable the chicken to breathe ambient air between collections of serial breath samples. Other approaches described in the literature such as insertion of a tracheal cannula (Scheid and Piiper, 1969) or the gluing of tubes into the nostrils (Itabisashi, 1981) were considered extreme from an animal welfare point of view and otherwise impractical or inappropriate. The nasal prong approach was also rejected due to the small size of the nostril openings in chickens.

A breakthrough came when we noticed that a cardboard tube from a roll of paper towel fitted neatly over the head of a chicken. From there, the idea developed into construction of a rigid helmet of the style used by early divers. We constructed helmets of different internal diameters and lengths from standard polyvinyl chloride (PVC) plumbing pipe and caps (Figure 1Go) for use with chickens of different age groups and, hence, sizes. The procedure for collecting a breath sample involved 2 people. One person held the chicken with one hand, and with the other hand, placed the helmet over the head and neck of the chicken, and then held it firmly against the shoulders and breast (Figure 1Go). The second person drew a 10-mL gas sample by opening a valve attached to an evacuated syringe, after a predetermined period measured by stopwatch. Then, the helmet was removed.


Figure 1
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  		Figure 1. Photograph of 22-d-old chicken with helmet in place for drawing re-breathed air into an evacuated syringe.

 
The choices of helmet size and the period of time that it was placed over the head of the chicken of a given age were based on previous trial and error for the time taken for re-breathing of air to increase the CO2 concentration in the enclosed space to at least 2% but not exceed 5%. Achievement of these limits was important, first, to ensure accurate analysis by mass spectroscopy (with total CO2 >1%) and, second, to avoid disruption to normal respiration by the chicken from excess CO2 in re-breathed air in the helmet.

Use of Helmets for Collecting Breath Samples for Measurement of 13CO2

Two substrates labeled with 13C, a stable isotope of carbon, were chosen for 2 experiments. The first substrate, 13C-octanoic acid, has been used to measure gastric emptying time for the liquid phase of ingested food in human subjects (Swart and van den Berg, 1998). The second substrate, corn, is naturally enriched in 13C through the metabolic pathway described by Hatch and Slack (1966), and has been used to examine solid-phase gastric emptying and starch hydrolysis in the small intestine of rats (Symonds et al., 1998). This substrate is readily available and economical compared with labeled probes normally used in clinical practice (Swart and van den Berg, 1998).

13CO2 in Expired Air Following Ingestion of 13C-Octanoic Acid

Following an overnight fast, 3 breath samples were taken at 3-min intervals from four 18-d-old chickens to establish the baseline 13C/12C isotope ratio. Each breath sample was taken 45 s after the helmet (40 mm in diameter, 100 mm long) was placed over the head of the chicken and held firmly against the shoulders to minimize loss of expired CO2. It was assumed that both isotopic forms of CO2 would diffuse at a similar rate under these circumstances; hence, any leakage should not have affected the ratio of 13CO2 to 12CO2 in the sample. Each chicken was then administered a gelatin capsule containing a weighed amount (approximately 95 mg) of vegetable oil containing 13C-octanoic acid (37.8 µg/mg of vegetable oil). That is, each chicken received approximately 3.6 mg of 13C-octanoic acid. For the first 2 birds, breath samples were taken at 10-min intervals for 1 h, then at 15-min intervals over the next hour, and then sampled at 30-min intervals for another 2 h. For the other 2 birds, breath samples were taken at 5-min intervals for 20 min, and then less frequently thereafter, for a total of 5 h. After each breath sample was taken, the chicken was allowed water but not food. Care was taken during handling and breath sampling to minimize stress on the chickens and disruption to normal breathing patterns. Breath samples were analyzed by isotope ratio mass spectrometry as described by Symonds et al. (2004).

13CO2 in Expired Breath Following Ingestion of Corn

Following an overnight fast, 3 breath samples were taken at 3-min intervals from three 25-d-old chickens to establish the baseline 13C/12C isotope ratio. Each breath sample was taken 35 s after the helmet (50 mm in diameter, 95 mm long) was placed over the head of the chicken. Each chicken was then administered 10 mL of canned corn (Edgell brand creamed corn, Simplot Australia Pty Ltd., Mentone, Victoria) via a disposable syringe fitted with a plastic tube that was inserted 4 cm into the esophagus. The canned corn was homogenized (IKA Ultra-Turrax T18, IKA Werke, Staufen, Germany) to pass through the syringe tip. Breath samples were taken at 15-min intervals for 2 h and then at 30-min intervals for another 2 h. After each breath sample was taken, the chicken was allowed water but not food. Breath samples were analyzed by isotope ratio mass spectrometry, as described previously.

Measurement of H2 and CH4 in Breath Samples from Chickens

Two experiments were conducted with chickens to determine if H2 and CH4 emerged in measurable concentrations in breath samples, and whether an oral dose of lactulose, a synthetic disaccharide used in diagnostic tests in human subjects, would result in an increase in H2 concentration in breath of chickens.

Breath samples were collected from 12 nonfasted 25-d-old chickens. Each breath sample was taken 30 s after the helmet (40 mm in diameter, 100 mm long) was placed over the head of the chicken. The helmet was removed and the chicken was returned to its metabolism cage. Chickens were allowed to eat and drink until fasting commenced in the evening of the following day, in preparation for further tests described in the section below.

The chickens were fasted overnight from d 26. Each chicken was weighed and breath tested to establish the predosing baseline levels of H2 and CH4, and was then administered with 5 mL of diluted lactulose solution via a disposable syringe fitted with a plastic tube that was inserted 4 cm into the esophagus. Each chicken was given approximately 130 mg of lactulose in 5 mL of water, which is equivalent to the dose given to human subjects to assess carbohydrate malabsorption. The lactulose solutions were made from Duphalac syrup (Solvay-Duphar B.V., Weesp, the Netherlands; 3.34 g of lactulose/5 mL) diluted in deionized water. Chickens were breath tested again at 3 h postdosing with lactulose. Breath samples were kept cool (0 to 4°C) to minimize gas diffusion during transport and storage before analysis. Hydrogen, CH4, and CO2 concentrations in breath were measured by gas chromatography as described by Symonds et al. (2004).

Data Analysis

Values for H2 and CH4 concentrations in breath samples were normalized to an assumed value of 5% for CO2 concentration in expired breath. The MEAN procedure in SAS (Windows version 9.1, SAS Institute Inc., Cary, NC) was used to calculate means, SD, CV, and ranges of concentrations of H2 and CH4 in breath samples.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Enrichment of 13C in breath CO2 (defined as delta over baseline, the increase in the ratio of 13C to 12C relative to the baseline determined for each chicken) following ingestion of 13C-octanoic acid is shown in Figure 2AGo. Peaks were observed 5 to 30 min postingestion. The enrichment of 13C in breath CO2 following ingestion of 13C-starch in corn is shown in Figure 2BGo. Peaks were observed 60 to 90 min postingestion.


Figure 2
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  		Figure 2. Analysis of breath samples from individual chickens. Enrichment of 13CO2 in breath following ingestion of A) a gelatin capsule containing 3.6 to 3.8 mg of 13C-octanoic acid dissolved in vegetable oil, and B) cooked corn kernel naturally enriched with 13C-starch. Delta over baseline is the increase in the ratio of 13C to 12C relative to the baseline ratio in each chicken before dosing. Each of the 4 curves represents results from an individual chicken. Panel C indicates breath H2 concentration (in ppm) in individual chickens fed a commercial broiler diet ad libitum, and then 2 d later from the same chickens fasted overnight immediately before and 3 h after oral dosing with lactulose (130 mg in 5 mL of water); panel D indicates breath CH4 (in ppm) in the same chickens as shown in panel C.

 
There was large between-bird variability in H2 and CH4 concentrations (in ppm) in breath samples from nonfasted chickens fed a commercial diet, and the same chickens 2 d later, just before and 3 h after dosing with lactulose (Figures 2C and 2DGo). Means, SD, CV, and ranges in concentrations of H2 and CH4 in breath samples are summarized in Table 1Go.


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  		Table 1. Means, SD, CV, and ranges of concentrations of H2 and CH4 in breath samples from 12 chickens
 
In 9 of 12 chickens studied, there was an increase in H2 concentration in the 3-h period following dosing with lactulose compared with fasted birds. There was no change in one chicken (number 4 in Figure 2CGo) and the 2 other chickens showed a decline (numbers 6 and 7 in Figure 2CGo) in H2 concentration. Overnight fasting resulted in a reduction in between-bird variability, possibly because of the reduced amount of fermentable material reaching the hindgut. Methane (up to 8 ppm) was detected in the breath of each of the 12 chickens at some stage during this experiment, either before or after dosing with lactulose (Figure 2DGo). In other experiments (data not shown), we failed to detect CH4 in breath of chickens from different hatchings but reared and fed in a similar way to those reported in this study.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results are consistent with observations in humans and other mammals in which 13C-octanoic acid is rapidly absorbed in the intestine, metabolized, and then excreted by the lungs (Tivey and Butler, 1999). Peak enrichment in chickens between 5 and 30 min is comparable with mice (Symonds et al., 1998) and considerably less than the 53-min delay observed in adult humans given a semisolid test meal containing sodium [13C1]-acetate to measure emptying of the liquid phase (Braden et al., 1995). Presumably, these different intervals of time to reach peak enrichment reflect the relative lengths of the gastrointestinal tract in mice, chickens, and adult humans.

The flattened, delayed peaks for 2 chickens (Figure 2AGo) indicate natural variation between birds in terms of gastric emptying time because no problems occurred during administration of the isotope or serial breath sampling. This was much quicker than the mean time of 153 min observed in human infants 7 to 16 mo of age with mean weight of 8.6 kg given a test meal made from maize flour (Weaver et al., 1995). Hiele et al. (1989) reported peaks in 13CO2 excretion at 3 and 5 h in healthy volunteers and patients with pancreatic disease, respectively, following consumption of a test meal made from corn starch suspended in water. The main difference between chickens and humans was the shorter time to peak enrichment of 13C in breath CO2 in chickens (60 to 90 min; Figure 2BGo), as was the case with 13C-octanoic acid (Figure 2AGo).

The degree of enrichment of 13CO2 in breath samples from chickens in both experiments was similar to that found with humans and other experimental animals dosed with these labeled substrates. The smooth transitions over time from zero enrichment to peaks and subsequent declines to baseline imply that the sampling procedures produced representative samples of expired breath (Figure 2A and 2BGo).

The patterns of recovery of the stable isotope in the form of 13CO2 in breath samples were comparable with those seen in humans, with the exception of the shorter time delay to peak enrichment in breath compared with adult humans. This is, perhaps, not surprising given the shortness of the gastrointestinal tract relative to body weight in chickens compared with mammals (Hill, 1971), more rapid transit of digesta in chickens than mammals (Vergara et al., 1989), and the greater metabolic rate of chickens compared with adult humans, as indicated by average body temperatures of 42 and 37°C, respectively. The similar recovery patterns suggest that there are no fundamental differences between avian and mammalian species in terms of the basic physiological and biochemical processes underpinning 13CO2 breath tests for gastric emptying and pancreatic function. Likewise, the appearance of measurable concentrations of H2 in breath collected from chickens, and the comparability of the breath profiles over time with that reported in other animals, indicate the potential for development of breath tests to aid in the diagnosis of maldigestion, malabsorption, and dysbacteriosis in commercial flocks.

These results imply that additional factors such as the rate of passage of digesta, proliferation of facultative anaerobes (Choct et al., 1996), and combinations of these may have contributed to the large coefficients of variation in breath H2 and CH4 (Table 1Go). In all chickens tested, gut microflora (presumably mainly in the ceca) were capable of producing H2 and CH4 from naturally occurring carbohydrate in the diet and a synthetic disaccharide (lactulose) administered orally. Fasting reduced the between-bird variability in breath H2 and CH4 concentrations. However, in other studies (data not published), we have observed the absence of CH4 in breath from chickens in different hatchings. Nevertheless, these results demonstrate the potential for breath tests to provide a simple way of tracking changes in metabolic activity of microflora in experimental animals before, during, and after administration of different dietary treatments and other therapies likely to alter the profile of gut microflora. Further work is required to validate that the H2-CH4 breath test can be used in this manner. Breath tests need to be done in conjunction with conventional measurements to characterize gut microbial profiles and fermentation patterns.

In conclusion, these preliminary experiments demonstrated that, using easily constructed PVC helmets and simple procedures, it is possible to collect breath samples from individual birds for measurement of concentrations of 13CO2, H2, and CH4, which form the basis for various diagnostic tests for intestinal dysfunction in humans. This opens the way for further studies to develop noninvasive tests applicable to chickens.


   ACKNOWLEDGMENTS
 
This research was supported by the Rural Industries Research and Development Corporation Chicken Meat Program (project SAR13A).


   FOOTNOTES
 
2 Current address: Veterinary Research Synergies Pty Ltd., East-wood SA 5063, Australia. Back

3 Current address: Women’s and Children’s Hospital, North Adelaide SA 5006, Australia. Back

Received for publication October 12, 2007. Accepted for publication May 11, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amarri, S., and L. T. Weaver. 1995. 13C-Breath tests to measure fat and carbohydrate digestion in clinical practice. Clin. Nutr. 14:149–154.[CrossRef][Web of Science][Medline]

Braden, B., S. Adams, L. P. Duan, K. H. Orth, F. D. Maul, B. Lembcke, G. Hor, and W. F. Caspary. 1995. The [13C] acetate breath test accurately reflects gastric emptying of liquids in both liquid and semisolid test meals. Gastroenterology 108:1048–1055.[CrossRef][Web of Science][Medline]

Choct, M., R. J. Hughes, J. Wang, M. R. Bedford, A. J. Morgan, and G. Annison. 1996. Increased small intestinal fermentation is partly responsible for the anti-nutritive activity of non-starch polysaccharides in chickens. Br. Poult. Sci. 37:609–621.[CrossRef][Web of Science][Medline]

Hatch, M. D., and C. R. Slack. 1966. Photosynthesis by sugarcane leaves: A new carboxylation reaction and the pathway of sugar formation. Biochem. J. 101:103–111.[Web of Science][Medline]

Heine, W. E., H. K. Berthold, and P. D. Klein. 1995. A novel stable isotope breath test: 13C-Labeled glycosyl ureides used as noninvasive markers of intestinal transit time. Am. J. Gastroenterol. 90:93–98.[Web of Science][Medline]

Hiele, M., Y. Ghoos, P. Rutgeerts, and G. Vantrappen. 1989. Starch digestion in normal subjects and patients with pancreatic disease, using a 13CO2 breath test. Gastroenterology 96:503–509.[Web of Science][Medline]

Hill, K. J. 1971. Structure of the alimentary tract. Pages 1–22 in Physiology and Biochemistry of the Domestic Fowl. D. J. Bell, and B. M. Freeman, ed. Academic Press, London, UK.

Itabisashi, T. 1981. Breath-by-breath analysis of expiratory gas concentration in chickens. Natl. Inst. Anim. Health Quart. Japan 21:42–51.

Robb, T. A., and G. P. Davidson. 1987. Two-hour lactose breath hydrogen test. J. Pediatr. Gastroenterol. Nutr. 6:481–482.[CrossRef][Web of Science][Medline]

Scheid, P., and J. Piiper. 1969. Volume, ventilation and compliance of the respiratory system in the domestic fowl. Respir. Physiol. 6:298–308.[CrossRef][Web of Science][Medline]

Swart, G. R., and J. W. O. van den Berg. 1998. 13C breath tests in gastroenterological practice. Scand. J. Gastroenterol. 33:13–18.[Medline]

Symonds, E. L., S. Kritas, T. Omari, and R. N. Butler. 2004. A combined 13CO2/H2 breath test can be used to assess starch digestion and fermentation in humans. J. Nutr. 134:1193–1196.[Abstract/Free Full Text]

Symonds, E. L., T. Omari, R. Butler, and G. Davidson. 1998. A novel technique for monitoring breath, applied to assessment of gastric emptying in the mouse. J. Gastroenterol. Hepatol. 13(Suppl.):A1111.[CrossRef]

Tivey, D. R., and R. N. Butler. 1999. Breath analysis - A key to understanding intestinal function. Recent Adv. Anim. Nutr. Aust. 13:45–52.

Vantrappen, G. R., P. J. Rutgeerts, Y. F. Ghoos, and M. I. Hiele. 1989. Mixed triglyceride breath test: A noninvasive test of pancreatic lipase activity in the duodenum. Gastroenterology 96:1124–1134.

Vergara, P., M. Jimenez, C. Ferrando, E. Fernandez, and E. Gonalons. 1989. Age influence on digestive transit time of particulate and soluble markers in broiler chickens. Poult. Sci. 68:185–189.[Web of Science][Medline]

Weaver, L. T., B. Dibba, B. Sonko, T. D. Bohane, and S. Hoare. 1995. Measurement of starch digestion of naturally 13C-enriched weaning foods, before and after partial digestion with amylase-rich flour, using a 13C breath test. Br. J. Nutr. 74:531–537.[CrossRef][Web of Science][Medline]

Wutzke, K. D., W. E. Heine, C. Plath, P. Leitzmann, M. Radke, C. Mohr, I. Richter, H. U. Gulzow, and D. Hobusch. 1997. Evaluation of orocaecal transit time: A comparison of the lactose-[13C, 15N]ureide 13CO2- and the lactulose H2– breath test. Eur. J. Clin. Nutr. 51:11–19.[CrossRef][Web of Science][Medline]





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