Respiration chambers (RC) have been used for studying the energy metabolism of animals and methane (CH₄) energy losses of ruminants for more than 100 years ^[1][2][3][28,30,31]. The principle of the RC technique relies on measuring CH₄ concentrations released from enteric fermentation (nasal and rectum) in gas samples and the total volume of air removed from the RC ^[1][4][5][6][4,28,32,33]. The chamber method uses only a few animals for continuous monitoring, usually over a course of 24 h periods, for 3–7 days ^[3][7][26,31]. Changes in O₂, CO₂, and CH₄ contents are calculated from the gas flow, and changes in gas concentrations between the air inlet and outlet are measured using gas analyzers, infrared (IR) photoacoustic monitors, or gas chromatography systems ^[8][9][24,34]. Respiration chambers provide an accurate reference method used for research purposes ^[7][26].

There are two types of RC: closed-circuit and open-circuit ^[10][35]. While the closed-circuit systems are these days almost never used, the open-circuit chambers are currently the most commonly used, with varying degrees of complexity ^[3][5][31,32]. Gas recovery is an essential routine maintenance task while performing RC experiments. Thresholds for the chamber temperature, relative humidity, CO₂ concentration, and ventilation rate are <27 °C, <90%, <0.5%, and 250–260 L/min, respectively ^[8][24].

Chambers need to be routinely calibrated and demonstrate gas recovery rates of close to 100%, both before and after each experimental deployment ^[11][12][36,37]. However, in practice, it is estimated that the average recovery value is 98.1% ^[13][5]. Respiration chambers have low animal-to-animal variations and good refinement in CH₄ measurements. They are suitable for studying the differences between treatments for mitigation strategies and are still regarded as the “gold standard” method for measuring individual animal CH₄ emissions ^[6][8][12][24,33,37]. However, RC use is technically demanding, and only a few animals can be monitored at the same time ^[14][38]. The chamber method has both high investment and labor costs ^[7][26]. Animals’ behavior is supposed to be affected by the artificial environment created by the method, and it is not suitable for free-ranging animals ^[1][28]. Nevertheless, RCs are the most appropriate for providing continuous and accurate data on air composition over an extended period of time ^[8][24].

The basic principle of the in vitro technique is incubating feed under gas-tight culture bottles involving natural rumen microbes under an anaerobic environment ^[1][8][24,28]. The gas measuring technique has been widely used for evaluating the nutritive value of feeds and simulating ruminal fermentation of feed and feedstuffs ^[5][15][32,39]. In this technique, feedstuffs are incubated for a specific time frame (2, 4, 8, 24, 48, 72, 96 and/or 144 h) with a mixture of reducing solution, buffer, and rumen fluid at 39 °C [4]. In the meantime, the total gas production and CH₄ are measured ^[1][28]. Blank samples with no feedstuffs are also run to correct for the amount of background gas produced.

The method requires access to fresh rumen fluid from fistulated animals, collected by esophageal tubing on intact animals or from slaughtered animals [4]. The method is ideal to screen different feedstuffs within a short time (1–4 weeks) in a controlled environment ^[1][4][4,28]. One way of determining the kinetic parameter of total gas production is by using the nonlinear curve fitting procedure in GenStat and SAS ^[8][16][24,40]. Syringes; Rusitec; closed vessel batch fermentation and fully automated systems have been used for CH₄ determination ^{[17][18][19][20]}[41,42,43,44]. The method allows as many replications in one batch to discern differences among treatments ^[1][28]. The result of this technique can serve as input to optimize larger and more expensive in vivo experiments [4]. However, the system can only simulate the ruminal fermentation of feed. Furthermore, under normal conditions, the system lacks to capture the thriving environment of rumen microorganisms in the tested feedstuffs ^[1][28].

The sulfur hexafluoride tracer method was first developed at Washington State University ^[21][45] and described in 1993–1994 by ^[22][19]. It is a widely used technique to measure enteric CH₄ emissions ^[12][37]. The technique provides a direct measurement of the CH₄ emission of individual animals ^[8][24]. The purpose of the SF₆ technique is to investigate how much CH₄ does the penned as well as free-ranging and grazing animals produce over a given period (24h feeding cycle) ^[3][4][23][4,31,46]. SF₆ is a non-toxic, physiologically inert, and stable gas that is easy to detect, even in minute amounts ^[4][5][24][4,32,47]. In addition, SF₆ gas mixes with rumen air in the same way as CH₄ ^[1][28].

The principle behind this method is that from the rumen, the SF₆ gas release rate is determined in order to calculate the CH₄ emission measurement ^[6][22][25][19,33,48]. The SF₆ gas release rate could be achieved by placing an SF₆ filled permeation tube in a 39 °C water bath. Once the release rate is known and reaches stability, the permeation tube will be placed in the rumen of the study animals ^[1][28].

The sampling apparatus consists of a small brass permeation tube placed in the rumen and a lightweight “yoke”, fitted with a collection PVC canister, a halter and capillary tubing in which an air-evacuated canister draws air at a slow and steady rate from near the animal’s nostrils ^{[1][4][5][21][23]}[4,28,32,45,46]. Eructated gas samples release both SF₆ and CH₄ from their nostrils, and some of this is sucked into the canister (along with air surrounding the animal) ^[4][23][4,46]. The ratio of CH₄:SF₆ in the canister is used to determine the daily CH₄ emission with each gas corrected for background concentration. The concentration of SF₆ and CH₄ in the canister is determined by gas chromatography ^[5][32], in conjunction with the pre-determined SF₆ permeation rate of the tubes ^[3][25][26][31,48,49]. Samples are advised to be taken over 24 h intervals, over a minimum period of five sequential days, with background air samples collected alongside animals at the same time ^[3][31]. The following equation is used to determine CH₄ emission using the SF₆ technique ^[6][33]. where [CH₄]c and [SF₆]c are the concentrations of CH₄ and SF₆ in the canister, respectively; while [CH₄] b and [SF₆] b are the CH₄ and SF₆ concentrations in the background air, respectively ^[6][33].

In theory, the SF₆ technique is recommended for grazing cattle involving large herds (n > 50), ^[5][27][32,50]. Furthermore, it can also be employed under more controlled conditions where the intake is measured and/or regulated ^[8][24]. The duration of collection of each sample is regulated by altering the length and/or diameter of the capillary tube ^[1][22][19,28].

Collecting adequate short-term breath data for measurements of emission are the essence of spot sampling methods ^[7][26]. The methods use spot measurement of exhaled CH₄ at milking or during feeding. Such methods are usually automated, non-invasive and non-intrusive, allowing a high throughput of animals ^[3][31]. Adequate data provide a repeatable estimate of emission rate and scale up from a short-term emission rate to CH₄ emissions for the whole day ^[5][32].

The idea of the sniffer method was first gestated by Garnsworthy et al. ^[28][51]. This method is based on short-duration continuous breath analysis of exhaled air from the feed troughs in automatic milking systems (AMS) or concentrate feeders (CF) ^[7][26]. To collect air eructed by animals during milking, a sample inlet is inserted in the feed manager of an autonomous milking system ^[28][51]. The sniffer method sample analysis is based on continuous sampling of air in the manager using data recorders to monitor CH₄ and CO₂ concentrations near the animal’s muzzle ^[6][33]. This method provides an estimate of total daily emissions by individual animals on-farm ^[3][31]. It also provides hundreds of repeated measurements over prolonged periods ^[12][37]. However, studies using the sniffer method have shown, a high between-animal coefficient of variation (CV) as compared to the RC and flux method ^{[7][29][30][31]}[26,52,53,54]. In addition, with this method, CH₄ and CO₂ concentrations are highly influenced by the distance of the animal’s head from the point of sampling, which is not an issue with total-air sampling ^[32][55].

GreenFeed^® (GF) is a patented, commercially available gas-flux quantification system (C-lock Inc., Rapid City, SD, USA) that combines an automatic feeding system with measures of CH₄, CO₂, airflow, and the detection of head position during each animal’s visit to the unit ^[7][8][33][24,26,56]. The GF method is based on the idea that many short-term CH₄ emission samples from an individual animal, taken several times throughout a day, can be aggregated to estimate an animal’s average daily CH₄ emission across several days/weeks/months ^[3][31]. The system measures CH₄ emissions from non-confined cattle and sheep and records short-term data (3–6 min) repeatedly over 24 h by attracting animals to the unit using a “bait” of pelleted concentrate ^[4][8][4,24]. This method uses a similar principle for measuring gas emissions as for respiration chambers (flux method) ^[7][26]. What makes the (GF) method special is that there are sensors that measure the concentration of CH₄ released from the animal’s mouth during the several minutes that the animal is feeding [4]. The head sensor also detects if the head of the cow is in the correct position before using the exhaled CH₄ concentration values for further calculations of the flux.

The GF system is embedded with automatic baiting, measurements of airflow and gas concentrations, electronics, communication devices, and a gas tracer device. Animal visits result in a feed reward and measurement of CH₄ emission after a specified time has elapsed between visits (determined by the investigator) ^[1][3][6][28,31,33]. Daily CH₄ emissions are estimated from multiple short-duration visits to the feed station over 1–2 weeks [4]. Daily CH₄ emission CH₄ (L/min) is calculated using the volumetric airflow rate (Fair (i)) adjusted to STP and corrected for the capture rate. where Cp(i) is the fractional capture rate of air at time i; [CH₄]c(i) and [CH₄]b(i) are the concentrations of captured gas (ppm) and background gas of CH₄ (ppm), respectively, at time i; and Fair(i) is the volumetric airflow rate (L/min) measured on a dry-gas basis at time i. ^[6][7][26,33]. The system provides comparable estimates to those produced both by RC and SF₆ techniques ^[8][34][24,57]. The measurements with sufficient duration (at least 3 min), and 30 observations were enough to obtain reliable CH₄ emission data, regardless of how many times per day the measurements were obtained ^[12][35][37,58]. For measuring CH₄ emissions from individual animals, GF is a more cost-effective method than both SF₆ and RC, both indoors and in pastures ^[3][12][31,37].

The principle of face mask (FM) for spot samplings of respiratory exchange and CH₄ emissions is based on animals trained to stay in sternal recumbency for 30 min measurement periods taken every 2–3 h with up to 7 measurements per day ^[36][37][59,60]. The method has been used to measure emissions from cattle, sheep, and goats ^[3][31]. The principle of this method is similar to RC in terms of measuring gas exchange and changes in the exhaled CH₄ concentration. It includes a mass flow controller, gas sampling unit, and CH₄ emission analyzer attached to each face mask, where gas measurements are corrected for differences in humidity, lag time, drift, and CH₄ emission (mL/min) for each period ^[38][61]. The FM method is comparatively cheaper and simpler than SF₆ or RC. Its mobility provides access to measure multiple locations to collect CH₄ emissions ^[39][62]. However, the number of measurements presented had a marked impact on animal behavior, as access to food and water was restricted during measurement periods. The FM method was also considered too laborious and interest in using the method to measure enteric CH₄ from ruminants has faded ^[38][61].

A portable accumulation chamber (PAC) system is essentially an airtight box without airflow ^[3][6][31,33]. The PAC consist of a clear polycarbonate box that has an opening at the bottom and that is sealed by achieving close contact with flexible rubber matting ^[8][24]. The method uses a portable air sampler and analyzer unit based on transform IR detection ^[5][32]. In this technique, PAC traps all exhaled gases during 2 h of sampling, during which oxygen is depleted, and a single measurement of CH₄ is taken at the end of the sampling ^[32][40][55,63].

One of the advantages of the PAC system is to facilitate easy access to emission measurements on grazing conditions, something not possible with immobile open-circuit chambers ^[8][24]. It allows for screening a large number of ruminants for an efficient CH₄ emission measurement ^[8][24]. However, the time of measurements relative to feeding and any postprandial changes in CH₄ emission is a potential source of variation in these measurements and thus, should be accounted for when the method is used ^[3][31].

3. Laser Technologies to Measure Enteric CH₄ Emission

3.1. The Laser CH

₄

Detector (Direct Measurements)

The use of lasers for gas detection has traditionally been used in environmental monitoring, air-quality monitoring, security, and health care ^[41][75]. A laser CH₄ detector (LMD) is used to monitor exhaled air CH₄ concentrations in the air between the laser device and the animal’s nose or mouth ^[12][42][37,76]. The LMD method is based on IR-absorption spectroscopy to establish the CH₄ concentration measurement ^[41][75]. It allows measurements of CH₄ emissions from the same animals repeatedly in their normal environments ^[6][33]. Measurements of CH₄ concentration are taken manually by a portable apparatus approximately 1–3 m from the animal ^[3][31]. The technique is similar to automated measurements of CH₄ concentration in exhaled air samples during milking or feeding, except here, measurements are taken from the animals’ nostrils ^[28][51]. The advantages of LMD over the traditional enteric CH₄ measurement techniques are that the LMD is a non-invasive, non-contact technique, with a fast response, and enables real-time measurements ^[41][75]. The author ^[41][75] concluded that LMD reflects a strong agreement between those recorded in the indirect open-circuit respiration calorimetric chambers ^[41][75]. However, the LMD technique is affected by factors, such as temperature, wind velocity, the proximity of other animals, humidity, and atmospheric pressure ^[6][12][33,37]. In a recent review by Sorg ^[43][77], it was suggested that the LMD method could be an alternative in situations where other methods are not suitable for use.

3.2. Open-Path Laser (Direct Measurements)

Open-path laser is a novel method for quantifying CH₄ emissions during feeding. It is currently been used to measure enteric CH₄ emissions from herds of animals ^[8][24]. The concept of this technique relies on lasers and wireless sensor networks that send beams of light from the herds of animals to an open-path tunable diode detector to analyze CH₄ from grazing animals by IR-absorption spectroscopy ^[44][45][78,79]. The laser comprises upwind and downwind paths for the predominant wind direction of the herd. The herd acts as a surface source or, when individual animals can be fitted with GPS collars, individual animals are treated as point sources. By combining the micrometeorological data, the method possibly measures whole-farm CH₄ emissions across several pastures ^[8][24]. However, wind directions, surface roughness, or periods of unfavorable atmospheric conditions (fog, rain, waves, heat, etc.) are a particular concern for the application of this technique ^[46][80].

4. Micrometeorological Methods

Micrometeorological methods are based on gas-flux measurements in the free atmosphere and the corresponding emission rates of animals ^[1][47][28,81]. The methods rely on concomitant measurements of wind velocity and CH₄ concentration ^[5][32]. For gas analysis, Fourier Transform Infrared (FTIR) spectroscopy is integrated into the system. However, there are differences in the measurement techniques and the calculation of emission rates. Some of the techniques available for emission measurements include mass balance, vertical flux, and Lagrangian dispersion analyses ^[47][81]. An advantage of these techniques is that it is possible to study animals within their normal production setting and the measurements can be made on a potentially large number of animals ^[47][81]. In addition, the methods can incorporate the measurement of footprint over larger areas ^[5][32]. It was confirmed that micrometeorological methods could give similar values of CH₄ emission compared to open-circuit respiration chambers ^[1][48][28,82]. It is, however, not possible to detect emissions from indoor-housed animals as well as from individual animals by using micrometeorological methods ^[6][33].