Portable Accumulation Chamber

NOTE: This version of Section 20 has been approved by the working group's Chair. Please be aware that further revisions may occur before final review and approval by the Board and ICAR members per the Approval of Page Process.

Introduction

In Australia and New Zealand an alternative method was developed for the short-term measurement of Methane Production Rate (MPR) of sheep using Portable Accumulation Chambers (PAC) during 1 hour without leading discomfort to the animals. Similarly to RC, CH₄ emissions recorded in PAC include gases from flatulence in addition to eructed and expired CH₄, but only during 1 hour. For a detailed comparison of the PAC and respiration chamber methods see Jonker et al. (2018). Currently PACs are mostly used for small ruminants, but there are trials underway for using larger cabins for large ruminants. In general the same protocols described here for small ruminants are also applied in the trials with large ruminants.

Eagle 2

Eagle 2 Gas analyser RKI Instruments, this device is often used to measure the following from the PACs:

• CH₄ (methane) concentration in ppm (ensure scale set to ppm by pushing the “range-shift" button until ppm comes up.

• CO₂ (carbon dioxide) concentration in %

• O₂ (oxygen) concentration in %

Calibration

Before the Eagle 2 can be used it needs to be calibrated, this requires specific gasses:

Methane (CH4) Calibration Gas

Methane Packaged in Steel Cylinders; Capacity: 103 Liters (3,6 Ft³)

40-004-(B) -(A) 100 ppm in Air 100ppm of Methane in Air

40-003-(B) -(A) 1.000 ppm in Air 1.000ppm of Methane in Air

Specialty mix

Calibration mix D cylinder (1.300 l), 5% CO₂, 16% O₂, 5.000ppm CH₄ (or some other specified mix)

The eagle 2 needs PALL Life Sciences Versapor® -1200 Membrane 25MM 1,2μm 100/Pk, circular paper filters for probe. The RKI part number for the filter bowl is RKI 80-0211RK Water Trap (bowl type) with 1641 fittings with pleated paper filter. Particle filter RKI 33-1200RK. This can be washed and thoroughly dried for re-use if required until replacement is possible.

Daily start up

Before starting the Eagle check the filters within the probe. These are in the circular region of the probe. Check the conical filter is dry and clean by unscrewing the middle section. Change the circular membrane paper filter at the start of each day & as needed throughout the day. Membrane paper filter is seated at the base of the probe closest to the black rubber tubing.

Reassemble the probe making sure all parts are sitting correctly. Turn machine on to warm up, for at least 20 minutes to allow the Eagle 2 to return to 0ppm Methane, 20,9% O₂ and 0% CO₂ before validations are carried out. If necessary, push demand zero by holding down the AIR demand button to zero the Eagle 2. This measurement is the “Fresh Air” measurement and should not be made within a building containing animals, rather it should be carried out in fresh air.

The Eagle 2 is checked against standard gases of known mix concentrations at the beginning and end of each day of recordings, using the 100ppm and 1.000ppm Methane in air gas bottles. All readings are recorded daily. Screw the regulator into the desired gas bottle. Insert the probe into the tube that is on the end of the regulator and open fully the tap that is on the regulator, allow reading to settle and record measurements on worksheet. The readings should be close to (within 20% of):

	Air	100ppm Methane mix	1.000ppm Methane mix
Methane (CH4)	0ppm	100ppm	1.000ppm
O2	20,9%	20,9%	20,9%
CO2	0%	0%	0%

If the readings shift too far from these levels, try again or the Eagle 2 may require servicing, recalibration, or sensor replacement.

Using the specialty mix

Open gas security valve. Open the valve on top of the gas bottle and turn the valve on and allow a little amount of gas to pass through the hose that is attached to the gas bottle, insert the probe into the hose (making sure the eagle is reading CH₄ = O, O₂=20,9, & CO₂= 0.

Another way to carry this operation out is fill a 3 Litre Tedlar Bag with the specialty gas. Once filled (don’t over fill the plastic bag) turn off the valve & remove gas pipe, place the probe from the eagle onto Tedlar bag, open valve and allow the eagle to settle & record the reading.

Record final readings of gas concentrations, usually the readings are 5.000 ppm methane and approximately 16% O₂ and 4,9% CO₂.

If these gas levels are not reached, remove probe, and allow meter to stand in fresh air for a few minutes, the display should show fresh air readings (20,9% O₂, 0% CO₂ and 0ppm Methane) if not demand zero using the “Air – Yes” button and try to measure the specialty mix gas a second time. If the Eagle 2 continues to read unacceptable gas levels the Eagle 2 may need to be serviced, recalibrated or need a sensor replacement. This Eagle 2 should not be used to take measurements, a different Eagle2 analyser must be used.

After known gas checks are completed allow the machine to return to a 0 ppm Methane before turning it off, this can take up to 20mins. You may have to push the demand zero button ("Air- Yes" button)  

These same gas calibration checks are to be done at the end of the day just prior to shutting the Eagle down in the same order as detailed above in daily start up procedures. These calibration values are recorded and if outside given parameters the machines are checked or recalibrated. The calibration data is not used in the calculations because the recorded data is rescaled as the first step before use in genetic evaluations.

Protocols on experimental farms

INIA - Uruguay - meat sheep

Below a protocol for using PACs to measure meat sheep from INIA in Uruguay.

The animal is placed in a sealed chamber (860-880 litres; for large animals/breeds, a larger chamber should be considered) for a known period of time, between 40 to 60 minutes, after at least three weeks of constant feeding in terms of quantity and type of feed. More than one measure (2 to 3) per animal is recommended, with a period no shorter than 7 days between measurements. The traits to evaluate would be the concentration of Oxygen, Carbon dioxide and Methane. On the measurement day with the animal placed into the chamber, CH₄, CO₂, and O₂ are recorded using a portable multi-gas detector (in parallel with a background estimation) every ten or twenty minutes. Air temperature and pressure will be also registered for the calculation of methane emission at standardized conditions. Multi-gas detector calibration, bump tests and chambers leak tests should be performed routinely. Sealing of the chamber is mandatory to guarantee isolation, which is highly recommended. Transparent chambers can be used to reduce stress, accounting for animal welfare. Records of body weight will be necessary to estimate actual gas volume in the chamber and to estimate methane intensity. Also, dry matter intake on the measurement day and previous days will be required to assess methane yield. When possible, animals can be off feed from one hour before the estimate, if extra handling is necessary and records of eaten feed and hour of last meal are available, they can stay on feed until the estimate (Robinson et al., 2020)

Data from each batch of methane measurements can be then transformed considering the body weight of the animal, the time between measures and start of measurement, the gas concentration inside and outside of the chamber, the temperature and atmospheric pressure, following the procedure described by Jonker et al. (2018).

Protocols on commercial farms

Norway - meat sheep

Below a protocol for using PACs from Norway for measuring meat sheep.

To use PAC on commercial farms, they are set in a truck. The truck is easier than a trailer to drive on icy roads, there is a possibility for heating, for cleaning and for carrying wastewater. 10 chambers are used, so that gases are measured in lots of 10 animals at a time. A hand-held Eagle2 instrument is used to capture accumulated 50 min gas emissions / consumption following a measurement protocol developed in New Zealand (Jonker et al., 2020). The sheep are placed in the PACs, the measurements last 50 minutes. Sheep are either fed fresh grass or grass silage and are required to be off feed for at least one and less than four hours prior to entering the chamber and are in addition weighed prior to measurement. Fifty-minute CH₄ concentration is converted to CH₄ g/hr.

The computation of CH₄, CO₂ and O₂ are realised as follows:

CH₄ / CO₂ emission and O₂ consumption are measured in gram/hour.

Conversion from weight to litres:  

• Litres CH₄/hour = ppm CH₄/hour * (1.146 - Weight * 1,01) / 1.000.000
• Litres CO₂/hour = ppm CO₂/hour * (1.146 - Weight * 1,01) / 100
• Litres O₂/hour = ppm O₂/hour * (1.146 - Weight * 1,01) / 100

1,01 is the density of a sheep; 1.146 is the air volume (in litre) in the PAC.

Converting mBar to kPa:

• gram_CH₄/hour = Litres CH₄/hour * (0,1 * Air Pressure, mBar) * 16,043 / (8,3145 * (Temp in °C + 273,15)
• gram_CO₂/hour = Litres CO₂/hour * (0,1 * Air Pressure, mBar) * 44,01 / (8,3145 * ( Temp in °C + 273,15)
• gram_O₂/hour = Litres O₂/hour * (0,1 * Air_Pressure, mBar) * 31,998 / (8,3145 * (Temp in °C + 273,15)

16,043 is the molar mass (g) of methane; 44,01 is the molar mass (g) of carbon dioxide; 31,998 is the molar mass (g) of oxygen; 8,3145 is the gas constant; 273,15 is to convert the temperature from °C to Kelvin.

New Zealand - sheep

Below a protocol used in New Zealand for sheep.

The sheep will be placed in the PAC for approximately 50 minutes and gas emissions will be measured at three time points using an Eagle 2 gas analyser (RKI instruments). Animals are weighed on the day of measurement and are grazed on known pasture covers for three days prior to measurement to allow determination of estimated dry matter intakes. Animals are handled as quietly as possible and measured once, using a random method of allocation to lots of 12 by sire.

• Lots are groups of animals allocated to be measured through the set of chambers at the same time, a lot consists of 12 animals.  For example, if there were a total of 96 animals to be measured in one day eight separate lots of 12 animals per lot would be randomly allocated.

• Allocations are completed using sire to allocate animals randomly to lots. Different allocations are carried out for each round of measurements ensuring animals are measured randomly across lots and days. e.g., a cohort of 156 animals would be split into 7 lots of 12 animals for the first day and 6 lots the following day.

Pre-Measurement

Animals to be measured through PAC are run as a single group along with their cohorts that will not be measured in PAC chambers. One week prior to measurement animals are drafted from the main group and run separately on pasture with desired feed levels. Three days prior to measurement all allocated animals are moved into a paddock of known area, pasture cover is above 2000kg/ha, paddock selection will depend on group size. On the day of measurement, an estimated pasture reading is obtained from the farmer and recorded.

Setting up the trailer

The chambers should be operated out of direct sunlight where possible to avoid heating up excessively while sheep are inside. If the chamber temperature gets above 30 °C let the animal out. The trailer should be as level as possible.

Day of Measurements

On the first day of measurements, animals are brought into yards and drafted into lots. The animals being measured on the first day are weighed and weights recorded and drafted into lots of 12, with only the morning lots (lots 1 – 3/4) animals to be measured retained off feed, with the balance for the afternoon lots put back on feed until approximately one hour before the afternoon session of measurements is due to start. The animals to be measured on day 2 are returned to the paddock. Day 2 animals are removed from feed on the day of their measurements, and pasture cover is re-estimated after they exit.

Measurements

Immediately prior to starting measurements, the air temperature and pressure outside the chambers should be recorded. Animals are located close to the chambers (trailer) set up and pushed into the chamber when the door to the chamber is open. The animals enters the chambers; the door is fastened closed. Upon the animal entering the chamber check that the pressure gauge on top of the chamber changes to reflect that a seal has been obtained. The first reading is taken immediately after the animals enter by inserting the Eagle 2 probe into the gas valve on the top of the chamber and opening the valve. After 30 seconds the reading should have stabilized and is recorded using a data recording on the XR. The values should be: 0ppm CH₄; 20,9% O; 0% CO_2.

Make sure the gas valve is closed before the probe is removed. The order of all animals and their IDs are recorded, and the “0” time measurements are taken for each animal in order of loading.

The methane value for these time measurements should be zero, if they are not zero it indicates that the chambers have not been suitably cleaned out between lots. If they are below this value, measurements can continue, but additional time should be allowed for the next cleaning period and a couple of chambers checked before any animals are loaded.

Animals are left undisturbed for 25 minutes from the first reading before a second measurement is taken. Ensure sufficient time is allowed for fresh air to enter the Eagle between measurements (3 – 4 minutes) when starting up the Eagle or leave the Eagle running. Make sure the Eagle is zero before starting second & third measurements.

The methane values for the second measurement should be at least 200ppm. If the measurement is between 100ppm and 200ppm, there may be an issue which should be checked and noted. To determine if the measurement was done correctly check the following things:

• Check that the chambers are sealed properly, and confirm reading, noting on the XR that it was a true measurement.  
• Check the door is closed and water is still in the water traps.  
• Check Eagle 2 is working properly  
• Check the animals have been fed to the required levels if there is more than two in the lot at this level.  

25 minutes after the second reading, a third reading is taken. Unless the CO₂ was above 2,75% at the second reading, then the third reading would be at the 40-minute mark. If the second measurement is above 2% CO2, the animal should be monitored at the 35-minute mark. If levels are above 5%, release the animal. If more than 2 or 3 in a lot move to 35-minute rounds, see early release of animals below.

After the third reading 50 minutes from the start, the door is unfastened, and the animal is released from the chamber. Before releasing animals inform any people in the surrounding area that the animals are being released, and check that the pathway is clear for animals to return to the pens. The chamber is then washed down with the water blaster.

Early release of animals

Any animals that have a CO₂ reading greater than 2% at their second reading need to be monitored and have an extra measurement made 10 minutes afterwards. If CO₂ rises above 5% at any point during the measurement time, the animal is immediately released.

If an animal does not settle in the first 10 minutes, or their behaviour is affecting others, the animal is released early, and a note is made on the PAC chamber recording sheet.

If an animal is showing visible agitation (hyperventilation or panting in the chamber) a final measurement is made, and the animal is released. An increase in frequency above 50 breaths per minute can be considered as panting but be aware that the other animals may become stressed when left on their own. Instead of releasing the stressed animal, consider opening the door a bit.

If an animal is released early a note on reason must be made.

Measurements for each Lot

Sheep spend approximately 50 minutes in PAC, while gas measurements are being taken.

• LWt = Live weight recorded before PAC measurements
• Air temperature is recorded in degrees celcius
• Air pressure is recorded in hectopascals (hPA) Note 1hPa = 1mb

Gas measurements are recorded at start, midpoint and for each individual recorded on XR. CH₄ in ppm and CO₂ and O₂ in %. Calculate litres/day (l/d) for each time period (1 = start to midpoint; 2 = midpoint to end, 3 = start to end).

For time period 3:

• CH₄ l/d = (End CH₄ – Start CH₄) / (End Time – Start Time) * (cVol - LWt * 1,01) / 1.000.000

• CO₂ l/d = (End CO₂ – Start CO₂) / (End Time – Start Time) * (cVol - LWt * 1,01) / 100

• O₂ l/d = = (Start O₂ – End O₂) / (End Time – Start Time) * (cVol - LWt * 1,01) / 100

Vol = 827 litres for mark 1 chambers, 853 for mark 2 chambers, and 1.146 for Mark 3 chambers. The volume and mark number of the chambers are written on them. The type of chambers used should be recorded on the daily recording sheet.

Convert L/d to grams/d:

Similar to computing g/hour.

• CH₄ g/d = CH₄ l/d * (Pressure(hPa) * 0,1) / (8,3145 * (Temperature(⁰C) + 273,15)) * 16,043
• CO₂ g/d = CO₂ l/d * (Pressure(hPa) * 0,1) / (8,3145 * (Temperature(⁰C) + 273,15)) * 44,01
• O₂ g/d = O₂ l/d * (Pressure(hPa) * 0,1) / (8,3145 * (Temperature(⁰C) + 273,15)) * 32

16,043 is the molar mass (g) of methane; 44,01 is the molar mass (g) of carbon dioxide; 31,998 is the molar mass (g) of oxygen; 8,3145 is the gas constant; 273,15 is to convert the temperature from °C to Kelvin.

Cleaning of the chambers

Between groups

The chambers have a gas extraction vacuum fan that is used to remove all residual gases from each chamber. This has to be done prior to the entry of the next animal to be measured.  

To operate the waste gas extraction fan, turn on the power once the final reading has been taken & open the door on the first chamber, let the animal out of chamber leave the door open and turn on red tap at top of chamber. Leave on for a minimum of 30 seconds and make sure tap is closed when loading the next lot.

In addition to removing the gases the chamber is also hosed out with a water blaster in preparation for the next animal. There is a drainage system at the rear of the chamber where water and waste drains from, plus any large amounts of waste are removed with a small spade.

After completing the trials for the day

Wash down and clean all chambers, leave the gas taps open to prevent condensation collecting in the tap. Complete three gas validations as done at the beginning of the day and record results. All chambers are checked as being empty.

Trait definition for meat sheep

GHG emissions can be expressed as raw outputs, in grams per day, or in relation to feed intake or body weight. Different indicators have been proposed and used to express GHG emissions.  

Ratio indicators:

• CH₄ or CO₂ yields express gas emissions in relation to dry matter intake: CH_{4 yield} = CH₄ / DMI
• CH₄ or CO₂ intensities express gas emissions in relation to live weight: CH_{4 intensity} = CH₄ / liveweight

Residual CH₄:

Similarly to residual feed intake, residual methane emissions (RME) has been proposed as an indicator of methane emissions.

In meat sheep, residual methane can be obtained as the residual from the following equation:

• Daily methane = µ + a × BW^0.75 + b x DMI + CG + RME (from Smith et al., 2021)

BW and DMI are body weight and dry matter intake, respectively, and have to be recorded over the same period as gas emissions. CG is the contemporary group.

Genetic evaluations

To decrease GHG emissions, selection programmes can use different models for the genetic analyses. The table below shows the models used by different analyses of GHG emissions criteria.

List of fixed and random effects used to analyse GHG emissions criteria.

Trait name	Species (DS, MS, DG)1	Fixed effects	Random effects	Notes
Gram CH4 / CO2  /O2 per hour	MS	Flock, age, lot (pen / trial), sex, birth type, dam age, live weight (fixed regression), age at measurement (fixed regression) birthdate deviation from average, rearing rank, age of dam	Animal

¹Species are defined as Dairy Sheep (DS), Meat Sheep (MS) and Dairy Goat (DG).

Genetic parameters

Genetic parameters obtained from the previously described protocols and using models presented in the above table are given in the below table.

Genetic parameters for GHG emissions traits estimated in small ruminants.

Traits	Species (DS, MS, DG)1	Heritability	Genetic standard deviation	Coefficient of variation (%)	Notes
CH4 (gram per hour)	MS (adult ewes)	0,18			SMARTER, Jette Jakobsen, NSG (Jakobsen et al., 2022)
CO2 (gram per hour)	MS (adult ewes)	0,31			SMARTER, Jette Jakobsen, NSG (Jakobsen et al., 2022)
CH4 (gram per day)	MS (wool)	0,23			Marques et al., 2022
CO2 (gram per day)	MS (wool)	0,27			Marques et al., 2022
O2 (gram per day)	MS (wool)	0,26			Marques et al., 2022
CH4 g/d	MS	0,19 (0,05)			Jonker et al., 2018
CO2 g/d	MS	0,19 (0,05)			Jonker et al., 2018
CH4 / (CH4 + CO2) (mol/mol)	MS	0,24 (0,05)			Jonker et al., 2018

¹Species are defined as Dairy Sheep (DS), Meat Sheep (MS) and Dairy Goat (DG).