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Section 20 – Methane Emission for Genetic Evaluation

2026-02-18T09:40:26Z

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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]].

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== Introduction ==
Increases in milk production through management and genetics have substantially improved feed efficiency and decreased costs per unit of product over recent decades. However, dairy systems are also associated with environmental costs (Baskaran et al., 2009<ref>Baskaran, R., Cullen, R., and Colombo, S. 2009. Estimating values of environmental impacts of dairy farming in New Zealand, New Zealand J. Agric. Res. 52: 377-389, DOI: 10.1080/00288230909510520.</ref>), with methane (CH4) emissions associated with rumen microbial fermentation being both an important contributor to global greenhouse gas (GHG) emissions, as well as an avoidable loss of energy that could otherwise be directed into milk production. The livestock sector is responsible for 14.5% of the global GHG (Gerber et al., 2013<ref>Gerber, P.J., Steinfeld, H., Henderson, B., Mottet, A., Opio, C., Dijkman, J., Falcucci, A. , and Tempio, G. 2013. Tackling climate change through livestock – A global assessment of emissions and mitigation opportunities. Food and Agriculture Organization of the United Nations (FAO), Rome.</ref>); dairy cattle account for 18.9% of these emissions, mainly in the form of enteric CH4 emissions (van Middelaar et al., 2014<ref>Van Middelaar, C.E., Dijkstra. J., Berentsen. P.B.M.. and De Boer. I.J.M. 2014. Cost-effectiveness of feeding strategies to reduce greenhouse gas emissions from dairy farming. J. Dairy Sci. 97:2427–2439.</ref>).

Methane is a greenhouse gas with a global warming potential 28 times that of CO2 (Myhre et al., 2013<ref>Myhre, G., Shindell, D., Bréon, F., Collins, W., Fuglestvedt, J., Huang, J., Koch, D., Lamarque, J., Lee, D., Mendoza, B., and Nakajima ,T. 2013. Anthropogenic and Natural Radiative Forcing. T.F. Stocker, D. Qin, G.-K. Plattner, M. Tignor, S.K. Allen, J. Boschung, A. Nauels, Y. Xia, V. Bex, and P.M. Midgley, ed. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA.</ref>). Methane from ruminant livestock is generated during microbial fermentation in the rumen and hindgut (enteric CH4), and from decomposition of manure. Enteric CH4 contributes 80% of CH4 emissions by ruminants, and manure decomposition contributes 20%. Enteric CH4 accounts for 17% of global CH4 emissions and 3.3% of total global greenhouse gas emissions from human activities (Knapp et al., 2014<ref>Knapp, J.R., Laur, G.L., Vadas, P.A., Weis,s W.P., and Tricarico, J.M. 2014. Invited review: Enteric methane in dairy cattle production: Quantifying the opportunities and impact of reducing emissions. J. Dairy Sci. 97:3231-3261.</ref>). There is, therefore, a significant research interest to find ways to reduce enteric CH4 emissions by ruminants.

Ruminant animals have a digestive system to digest plant materials efficiently. Like most mammals, ruminants lack the cellulase enzyme required to break the beta-glucose linkages in cellulose, but they play host to diverse populations of rumen microbes that can digest cellulose and other plant constituents. When rumen bacteria, protozoa and fungi ferment carbohydrates and proteins of plant materials, they produce volatile fatty acids, principally acetate, propionate and butyrate. High fibre diets favour acetate synthesis. Synthesis of acetate and butyrate are accompanied by release of metabolic hydrogen, which, if allowed to accumulate in rumen fluid, has negative effects on microbial growth, and feed digestibility (Janssen, 2010<ref>Janssen, P.H. 2010. Influence of hydrogen on rumen methane formation and fermentation balances through microbial growth kinetics and fermentation thermodynamics. Anim. Feed Sci. Technol. 160:1–22. doi:10.1016/j.anifeedsci.2010.07.002.</ref>). Rumen Archaea are microorganisms that combine metabolic hydrogen with CO2 to produce CH4 and water. Archaea play a vital role, therefore, in protecting the rumen from excess metabolic hydrogen, and the CH4 they produce is an inevitable product of rumen fermentation.

A number of CH4 phenotypes have been defined (Hellwing et al., 2012<ref>Hellwing, A.L.F., Lund, P., Weisbjerg, M.R., Brask, M., and Hvelplund. T. 2012. Technical note: test of a low-cost and animal-friendly system for measuring methane emissions from dairy cows. J. Dairy Sci. 95:6077–85. doi:10.3168/jds.2012-5505.</ref>); the most widely used is CH4 production (MeP) in liters or grams per day.

The CH4 production trait is highly correlated with feed intake (Basarab et al., 2013<ref>Basarab, J., Beauchemin, K., Baron, V., Ominski, K., Guan, L., Miller, S., and Crowley, J. 2013. Reducing GHG emissions through genetic improvement for feed efficiency: Effects on economically important traits and enteric methane production. Animal, 7(S2):303-315. doi:10.1017/S1751731113000888</ref>; De Haas et al., 2017<ref name=":0">de Haas, Y., Pszczola, M., Soyeurt, H., Wall, E., and Lassen, J. 2017. Invited review: Phenotypes to genetically reduce greenhouse gas emissions in dairying. J. Dairy Sci. 100:855-870.</ref>) and, thereby, with the ultimate breeding goal trait: milk production in dairy cattle. The economic value of daily dry matter intake and associated methane emissions in dairy cattle showed that increasing the feed performance estimated breeding value by one unit (i.e. 1 kg of more efficiently converted DMI during the cow’s first lactation) translates to a total lifetime saving of 3.23 kg in DMI and 0.055 kg in methane (Richardson et al., 2019<ref>Richardson, C., Baes, C., Amer, P., Quinton, C., Martin, P., Osborne, V., Pryce, J.E., and Miglior, F. 2020. Determining the economic value of daily dry matter intake and associated methane emissions in dairy cattle. Animal 14:171-179. doi:10.1017/S175173111900154X</ref>). Feed Performance was defined as a 1 kg increase in more efficiently used feed in a first parity lactating cow. These results show not only the relation between DMI and CH4 production, but also the economic relationship between these traits. Persistency of lactation was found to be positively associated with increased feed efficiency and decreased methane production and intensity. Feed efficiency was associated with lower methane intensity. Feed efficiency and methane emissions can be improved by selecting for dairy cattle that are smaller and have increased persistency of lactation. Efficiency and methane emissions can be further improved by improved management of body condition score and by extending lactations beyond the conventional 305-day length (Seymour, 2019<ref>Seymour, D.J. 2019. Feed Efficiency Dynamics in Relation to Lactation and Methane Emissions in Dairy Cattle. PhD thesis, The University of Guelph, Canada.</ref>). According to Ellis et al. (2007)<ref>Ellis, J.L., Kebreab, E., Odongo, N.E., McBride, B.W., Okine, E.K., and France, J. 2007. Prediction of methane production from dairy and beef cattle. J. Dairy Sci. 90:3456–3466.</ref>, DMI predicted MeP with an R2 of 0.64, and ME intake (MJ/d) predicted MeP with an R2 of 0.53 for dairy cattle. AlternativePhenotype definitions include CH4 intensity (MeI), which is defined as liters or grams of CH4 per kg of milk, and CH4 yield (MeY), which is defined as liters or grams of CH4 per kg of dry matter intake (DMI) (Moate et al., 2016<ref>Moate, P.J., Deighton, M.H., Williams, S.R.O., Pryce, J.E., Hayes, B.J., Jacobs, J.L., Eckard, R.J., Hannah, M.C. and Wales, W.J., 2016. Reducing the carbon footprint of Australian milk production by mitigation of enteric methane emissions. Anim. Prod. Sci. 56:1017-1034.</ref>). Residual CH4 production (RMP) is calculated as observed minus predicted CH4 production (Herd et al., 2014<ref>Herd, R.M., Arthur, P.F., Bird, S.H., Donoghue, K.A., and Hegarty, R.S. 2014. Genetic variation for methane traits in beef cattle. In: Proc. 10th World Conference on Genetic Applied to Livestock Production (WCGALP), 17-22 August, 2014. Vancouver, Canada.</ref>, Berry et al., 2015<ref>Berry, D.P., Lassen, J., and de Haas, Y. 2015. Residual feed intake and breeding approaches for enteric methane mitigation In: Livestock production and climate change. P.K. Malik, R. Bhatta, J. Takahashi, R.A. Kohn, and C.S. Prasad, ed. CABI, Oxfordshire, UK. . Pages 273-291</ref>), with predicted values based on factors such as milk production, body weight and feed intake. At the moment, it is not obvious which of these phenotypes to use; but, it is important to monitor associations between the chosen CH4 phenotype and the other important traits in the breeding goal (e.g. production, fertility, longevity) to avoid unfavorable consequences. Berry and Crowley (2012)<ref>Berry, D.P., and Crowley, J.J. 2012. Residual intake and body weight gain: A new measure of efficiency in growing cattle, J. Anim. Sci. 90:109–115, <nowiki>https://doi.org/10.2527/jas.2011-4245</nowiki></ref> describe advantages and limitations of ration traits. For example, because feed efficiency traits are a linear combination of other traits it is not recommended to include them in an overall total merit index, which is a clear limitation. For all applications it is necessary to measure the CH4 emission of each animal individually. These guidelines are intended to make the right choices for this.

Whilst diet changes and feed additives can be effective mitigation strategies for CH4 emissions (Beauchemin et al., 2009<ref>Beauchemin, K.A., McAllister, T.A., and McGinn, S.M. 2009. Dietary mitigation of enteric methane from cattle. CAB Rev.: Perspect. Agric., Vet. Sci., Nutr. Nat. Res. 4:1–18.</ref>; Martin et al., 2010<ref>Martin C., Morgavi, D.P., and Doreau, M. 2010. Methane mitigation in ruminants: from microbe to the farm scale. Animal 4:351–365.</ref>; Hristov et al., 2013<ref>Hristov, A.N., Oh, J., Firkins, J.L., Dijkstra, J., Kebreab, E., Waghorn, G., Makkar, H.P.S., Adesogan, A.T., Yan,g W., Lee, C., Gerber, P.J., Henderson, B., and Tricarico, J.M. 2013. Special topics - Mitigation of methane and nitrous oxide emissions from animal operations: I. A review of enteric methane mitigation options. J. Anim. Sci. 91:5045–5069.</ref>), their effects depend on the continued use of a particular diet or additive and there have been issues with the rumen microbiomes adapting to additives. Rumen bacterial communities are highly dynamic after a diet switch and did not stabilize within 5 wk of cows grazing pasture (Bainbridge et al., 2016<ref>Bainbridge, M.L., Saldinger, L.K., Barlow, J.W., Alvez, J.P., Roman, J. Kraft, J. 2016. 1609 Rumen bacterial communities continue to shift five weeks after switching diets from conserved forage to pasture. J. Anim. Sci. 94, suppl_5:783, <nowiki>https://doi.org/10.2527/jam2016-1609</nowiki> (abstr.)</ref>). In contrast, breeding for reduced CH4 emissions should result in a permanent and cumulative reduction of emissions (Wall et al., 2010<ref>Wall, E., Simm, G., and Moran, D. 2010. Developing breeding schemes to assist mitigation of greenhouse gas emissions. Animal 4:366-376.</ref>). Several studies have shown that CH4 emissions by ruminants have a genetic component, with heritability in the range 0.20 – 0.30 (de Haas et al., 2011<ref>de Haas, Y., Windig, J.J., Calus, M.P.L., Dijkstra, J., de Haan, M., Bannink, A., and Veerkamp, R F. 2011. Genetic parameters for predicted methane production and potential for reducing enteric emissions through genomic selection. J. Dairy Sci. 94:6122–6134.</ref>; Donoghue et al., 2013<ref>Donoghue K.A., Herd, R.M., Bird, S.H., Arthur, P.F., and Hegarty, R F. 2013. Preliminary genetic parameters for methane production in Australian beef cattle. In: Proceedings of the Association for the Advancement of Animal Breeding and Genetics, 20-23 October 2013, Napier, New Zealand, pp. 290–293.</ref>; Pinares-Patiño et al., 2013<ref>Pinares-Patiño C.S., Hickey, S.M., Young, E.A., Dodds, K.G., MacLean, S., Molano, G., Sandoval, E., Kjestrup, H., Harland, R., Pickering, N.K., and McEwan, J.C. 2013. Heritability estimates of methane emissions from sheep. Animal 7: 316–321.</ref>, Kandel et al., 2014A<ref>Kandel, P.B., Vanderick, S., Vanrobays, M.L., Vanlierde, A., Dehareng, F., Froidmont, E., Soyeurt, H., and Gengler, N. 2014A. Consequences of selection for environmental impact traits in dairy cows. Page 19. (<nowiki>http://orbi.ulg.ac.be/bitstream/2268/164402/164401/NSABS162014_poster_Purna_abstract.pdf</nowiki>) I:n Proc. 19th National symposium on applied biological sciences, Gembloux, Belgium.</ref>, B<ref>Kandel, P.B., Vanderick, S., Vanrobays, M.L., Vanlierde, A., Dehareng, F., Froidmont, E., Soyeur,t H., and Gengler, N. 2014B. Consequences of selection for environmental impact traits in dairy cows. In: 10th World Congress on Genetics Applied to Livestock Production (WCGALP), 17-22 August, 2014. Vancouver, Canada.</ref>; Lassen and Lovendahl, 2016<ref>Lassen, J., and Løvendahl, P. 2016. Heritability estimates for enteric methane emissions from Holstein cattle measured using noninvasive methods. J. Dairy Sci. 99:1959-1967.</ref>; López-Paredes et al. 2020<ref>Lopez-Paredes, J., Goiri, I., Atxaerandio, R., García-Rodríguez, A., Ugarte, E., Jiménez-Montero, J.A., Alenda, R and González-Recio, O. 2020. Mitigation of greenhouse gases in dairy cattle via genetic selection (i): Genetic parameters of direct methane using non-invasive methods and its proxies. J. Dairy Sci. 103.</ref>). Breeding for reduced CH4 emissions, alone or together with other mitigation strategies, could therefore be effective in reducing the environmental impact of cattle farming and, possibly, also in increasing feed efficiency. Such a breeding scheme would require, as a fundamental starting point, accurate measures of individual CH4 emissions on a large scale.

Several techniques have been developed for the measurement of CH4 emissions from ruminants, with varying degrees of accuracy (see reviews by Cassandro et al., 2013<ref>Cassandro, M., Mele, M., Stefanon, B.. 2013. Genetic aspects of enteric methane emission in livestock ruminants. Italian J. Anim. Sci. 12:e73: 450-458.</ref> and Hammond et al., 2016A<ref>Hammond, K.J., Crompton, L.A., Bannink, A., Dijkstra, J., Yáñez-Ruiz, D.R., O’Kiely, P., Kebreab, E., Eugenè, M.A., Yu, Z., Shingfield, K.J., Schwarm, A., Hristov, A.N., and Reynolds, C.K. 2016A. Review of current in vivo measurement techniques for quantifying enteric methane emission from ruminants. Anim. Feed Sci. Technol. 219:13–30. doi:10.1016/j.anifeedsci.2016.05.018.</ref>), but routine individual measurements on a large scale (a requisite for genetic selection) have proven to be difficult to obtain and expensive to measure (Pickering et al., 2015<ref>Pickering, N.K., Oddy, V.H., Basarab, J.A., Cammack, K., Hayes, B J., Hegarty, R.S., McEwan, J.C., Miller, S., Pinares, C., and de Haas, Y. 2015. Invited review: Genetic possibilities to reduce enteric methane emissions from ruminants. Animal 9:1431-1440.</ref>; Negussie et al., 2016<ref>Negussie E., Lehtinen, J., Mäntysaari, P., Liinamo, A-E., Mäntysaari, E., and Lidauer, M.. 2016. Non-invasive individual methane measurements in dairy cows using photoacoustic infrared spectroscopy technique. 6th Greenhouse Gases Animal Agriculture Conference (GGAA2016) 14-18 February 2016. Melbourne, Australia. Abstract. p62.</ref>). Therefore, identifying proxies (i.e. indicators or indirect traits) that are correlated to CH4 emissions, but which are easy and relatively lowcost to record on a large scale, would be a welcome alternative. Proxies might be less accurate but could be measured repeatedly to reduce random noise and in much larger populations.

These guidelines are highly indebted to Garnsworthy et al. (2019)<ref name=":1">Garnsworthy, P.C. Difford, G.F. Bell, M.J. Bayat, A.R. Huhtanen, P. Kuhla, B. Lassen, J. Peiren, N. Pszczola, M; Sorg, D. Visker, M.H., and Yan, T. 2019 Comparison of Methods to Measure Methane for Use in Genetic Evaluation of Dairy Cattle. Animals 9:837, 12p.</ref>. In this paper the methods to measure CH4 are compared with special emphasis to the genetic evaluation of dairy cattle.

== Disclaimer ==
The fact that specific device manufacturers are mentioned in these guidelines is in no way an endorsement of the devices or their accuracy by ICAR.

== Scope ==
A variety of technologies are being developed and employed to measure CH4 emissions of individual dairy cattle under various environmental conditions, as is evidenced by frequent reviews (Storm et al., 2012<ref>Storm, I.M., Hellwing, A.L.F., Nielsen, N.I., and Madsen, J. 2012. Methods for measuring and estimating methane emission from ruminants. Animals 2:160-183.</ref>; Cassandro et al., 2013<ref>Cassandro, M., Mele, M., Stefanon, B.. 2013. Genetic aspects of enteric methane emission in livestock ruminants. Italian J. Anim. Sci. 12:e73: 450-458.</ref>; Hammond et al., 2016A<ref>Hammond, K.J., Crompton, L.A., Bannink, A., Dijkstra, J., Yáñez-Ruiz, D.R., O’Kiely, P., Kebreab, E., Eugenè, M.A., Yu, Z., Shingfield, K.J., Schwarm, A., Hristov, A.N., and Reynolds, C.K. 2016A. Review of current in vivo measurement techniques for quantifying enteric methane emission from ruminants. Anim. Feed Sci. Technol. 219:13–30. doi:10.1016/j.anifeedsci.2016.05.018.</ref>; de Haas et al., 2017<ref>de Haas, Y., Pszczola, M., Soyeurt, H., Wall, E., and Lassen, J. 2017. Invited review: Phenotypes to genetically reduce greenhouse gas emissions in dairying. J. Dairy Sci. 100:855-870.</ref>). The first objective of the current guidelines is to review and compare the suitability of methods for large-scale measurements of CH4 output of individual animals, which may be combined with other databases for genetic evaluations. Comparisons include assessing the accuracy, precision and correlation between methods. Combining datasets from different countries and research centres could be a successful strategy for making genetic progress in this difficult to measure trait if the methods are correlated (de Haas et al., 2017<ref name=":0" />). Accuracy and precision of methods are important. Data from different sources need to be appropriately weighted or adjusted when combined, so any methods can be combined if they are suitably correlated with the ‘true’ value. The second objective of the current guidelines, therefore, is to examine correlations among results obtained by different methods, ultimately leading to an estimate of confidence limits for selecting individual animals that are high or low emitters (see also Garnsworthy et al., 2019<ref name=":1" />).

== Sub-sections ==
<div style="column-count:2">
:[[Section 20: Definition and Terminology |Definition and Terminology]]
:[[Section 20: Methane determining factors |Methane determining factors]]
:[[Section 20: Methane measuring methods|Methane measurements methods]]
:[[Section 20: Discussion of methods |Discussion of methods]]
:[[Section 20: Comparison of methods to measure methane |Comparison of methods to measure methane]]
:[[Section 20: Proxies |Proxies]]
:[[Section 20: Proxies Discussion|Proxies discussion]]
:
:[[Section 20: Merging and sharing data in genetic evaluations |Merging and sharing data in genetic evaluations]]
:[[Section 20: Activities|Activities]]
:[[Section 20: Recommendations|Recommendations]]
:[[Section 20: Conclusions |Conclusions]]
:[[Section 20: Beef|Beef]]
:[[Section 20: ICAR validation methane recording devices | ICAR validation methane recording devices]]

</div>

== Summary of Changes ==
{| class="wikitable sortable"
|+
!Date of change
! Nature of change
|-
|March 2020
|Draft from Feed & Gas WG put into standard template for ICAR Guidelines. Separate out EDGP database to become a standalone appendix.
|-
|April 2020
|Edits and acknowledgements added by Feed & Gas WG.
|-
|May 2020
|Approved by ICAR Board on 26th May subject to addition of disclaimer.
Disclaimer added as new chapter 2 - the fact specific device manufacturers are mentioned in these guidelines is in no way an endrosement of the devices or their accuracy by ICAR.
|-
|December 2023
|Creation of Methane Emission for Genetic Evaluation Wiki Page.
|-
|November 2025
|Creation of the Validation of methane recording devices at ICAR Test Centre
|}
==References==

Section 20: ICAR validation methane recording devices

2026-02-18T09:39:17Z

Bgolden: Bgolden moved page ICAR validation methane recording devices to Section 20: ICAR validation methane recording devices without leaving a redirect

== Introduction ==
ICAR validation ensures that a device (e.g. methane recording devices) meets manufacturer performance claims through ICAR-approved test plans conducted by a qualified ICAR Test Center. Successful validation confirms that the system can reliably deliver quality data when used correctly, leading to the award of an '''ICAR Certificate of Validation'''.

== Procedure with application for ICAR validation of methane recording and its claims ==
[[Section 11 – Testing, Approval and Checking of Measuring, Recording and Sampling Devices#Part 11C – Testing of Sensor Systems for ICAR Validation|The procedure for validation]] (Figure 1) starts with a request or claim by an applicant, which can be a manufacturer, another stakeholder or a combination of these, to the ICAR Secretariat. The applicant should apply through the application form available through the ICAR Secretariat and ICAR website. A complete application includes:

* General features of the applicant (name, addresses, physical location, contact person), to be provided filling an ICAR application form;
* Information on outsourced processes used by the applicant that are relevant for the evaluation;
* Methane recording name and type, additional ‘brand and type’ names;
* Purpose and measurement parameters;
* Where and how to be applied (laboratory, on-farm at-line, on-farm in-line);
* Mounting position or use;
* Specie(s) - i.e. cow, buffalo, sheep, goat, other;
* Peer reviewed publications;
* Reports of validation studies;
* Claim(s) to be validated ;
* Test type required (new device, testing a modification);
* Technical characteristics, drawings and photographs of device;
* Technical manual outlining functional processes and principles as well as software/firmware documentation.
* Installation procedure;
* User manual, including instructions on proper maintenance;
* Routine test or periodic check procedures for operators and service technicians.

Following the payment of a first part of the application fee by the applicant, the following steps will be taken:

* ICAR Secretariat will check the application form and related documentation.
* ICAR will recruit and appoint experts to form an ICAR Expert Panel. This Expert Panel reviews the application with the request/claim.
* ICAR, on recommendation of the ICAR Expert Panel will appoint an ICAR Test Centre to conduct the tests.
* The ICAR Expert Panel will formulate a test plan in consultation with the applicant and the ICAR appointed Test Centre.
* ICAR issues an umbrella contract and sends it to the applicant together with the test plan and the invoice for the full test fees.
* The ICAR test will be scheduled and conducted after the contract is signed and the test fees are paid in full. The ICAR Secretariat coordinates financial transactions between the applicant, the ICAR appointed Test Centre, and ICAR.
* For the test to begin, the applicant must send all the necessary devices and accessories to the ICAR appointed Test Centre. In addition, all manuals, documents, and procedures should be provided to the ICAR appointed Test Centre.
* Then the ICAR appointed Test Centre will conduct the tests. The ICAR appointed Test Centre is obliged to act according to the procedures laid down in the test plan and accompanying protocols. All details associated with the testing phase, including the test results, are kept strictly confidential. The ICAR appointed Test Centre provides periodic reports on tests in progress to the concerned ICAR Expert Panel.
* Upon completion of the test, the ICAR appointed Test Centre provides a draft report on the test results to the concerned ICAR Expert Panel through the ICAR Secretariat.
* After a 21-day review and comment period by the ICAR Expert Panel, the ICAR appointed Test Centre addresses any possible comments, prepares a final report and resubmits it to the ICAR Expert Panel through the ICAR Secretariat within 21 days of the receipt of the comments.
* After another 21-day review period, the ICAR Expert Panel provides its recommendation on validation output to ICAR Certification with a notification to the applicant.
* In case of a successful test for validation, ICAR will grant an ICAR certificate of validation to the applicant.
* All ICAR-validated claims are published on the ICAR website, including pictures of the device, year of validation, species and information on the certified claim.

[[File:Flowchart for ICAR validation.png|980x980px|Figure 1. Flowchart for ICAR validation|alt=Figure 1. Flowchart for ICAR validation|center|thumb]]

== Testing procedure for methane recording devices ==
The testing procedure consists of two stages: Firstly, the sniffer will be tested in the Air Quality Lab of [https://www.wur.nl/en.htm Wageningen University and Research] (Droevendaalsesteeg 3a, 6708 PB Wageningen, the Netherlands) and after the sniffer producing company and Wageningen University and Research agree on continuing the test procedure, the sniffer will be tested on farm at the innovation and research center [https://www.dairycampus.nl/nl/home.htm DairyCampus]; the official ICAR Test Centre (Boksumerdyk 11, 8912 CA Leeuwarden, the Netherlands).

=== Test in Air Quality Lab of Wageningen University and Research ===
At the Air Quality Lab known concentrations of CH4 and, if applicable, CO2 will be offered to the sniffers to validate the output of the sniffers. The offered CH4 concentrations are within a range of 100 ppm to 2,000 ppm and, if applicable, for CO2 within a range of 1,000 ppm to 10,000 ppm. The test procedure stipulates to start with the lowest concentration going to the highest concentration and back to also capture the capability of the sniffer to record sinking gas concentrations. At the lab, moisture sensitivity will also be tested for the sniffers. Here a known CH4 gas concentration is offered, and the level of relative humidity is increased in 5 steps from 0 ‘dry’ gas to 90-95%, which is the maximum amount. The recorded measures, the response time of the sniffer, the repeatability and the effect of moisture in the lab test will be documented and analyzed.

=== On farm test on DairyCampus ===
Field testing of the sniffers will be conducted on the DairyCampus farm. The barn in which the test will be conducted has a milking robot (DeLaval) as well as a [[Greenfeed SOP|GreenFeed unit]] (c-Lock Inc.) measuring the gas fluxes of CH4 and CO2. The sniffer will be installed for 3 months continuously at the milking robot. The repeatability of repeated measurements on the same cow on the same day, as well as the correlation with GreenFeed measurements on the same cow on the same day will be calculated and missing measurements will be documented. The ranking of the animals from low to high emitters will be compared.

Further, once per month the [[:File:Determination of carbon dioxide concentrations in-wageningen university and research LUNG METHOD.pdf|lung method]] will be applied to validate the output from the sniffers in the field. This is done by placing two gas collection bins next to the milking robot and collecting air samples from the same tube as the sniffers. We will collect samples at three different time points during the day. Each sample collected will be from an approximately 2-hour time period, in duplo (duplicate of air sampling at the same time). This collected gas will then be sent to the air-quality lab to be analyzed with gas chromatography (GC) to determine the concentrations of CH4 and CO2 in each bag. The results from this GC analysis will be compared to the average gas measurements from the sniffers over the same time period.

Section 10 – Identification Device Certification

2025-09-26T14:18:25Z

Bgolden: /* Procedure 6: Voluntary sampling of Identification Devices */

= Overview =
<div class="mw-collapsible mw-collapsed">
<div>
== Introduction ==
On June 22, 2007, the International Standards Organisation (ISO) appointed ICAR as the Registration Authority (RA) competent to register manufacturer codes used in the radio frequency identification (RFID) of animals in accordance with ISO 11784 and ISO 11785.

ICAR has administrative procedures in place for testing the conformance of RFID devices with respect to ISO 11784 and ISO 11785, and only ICAR-accredited test centres can conduct the ICAR certification testing. In addition, ICAR offers evaluations on various quality and performance features of livestock identification devices and transceivers that are tested for conformance with ISO 11784 and ISO 11785. A wide range of evaluations is also available for conventional plastic ear tags.

== Definitions and terminology ==
{| class="wikitable"
| colspan="2" |''Table 1. Definitions of terms used in these guidelines.''
|-
|'''Term'''
|'''Definition'''
|-
|RFID devices
|Animal identification devices (ear tags, leg tags, boluses, injectable transponders) using radio-frequency identification technology.
|-
|Conventional ear tags
|Visual ear tags used for animal identification that do not use RFID technology. These devices may or may not be machine readable (barcoded).
|-
|Registration Authority (RA)
|Authority appointed by ISO, competent to register manufacturer codes used in the radio frequency identification of animals in accordance with ISO standards 11784 and 11785.
|-
|Test centre
|ICAR-accredited laboratory that carries out tests on animal identification devices.
|-
|Registration
|The granting of shared/unshared manufacturer codes and unique product codes as defined in ISO 11784.
|-
|Manufacturer code
|A 3-digit number granted by ICAR to a manufacturer.
|-
|Certification
|ICAR service additional to the registration of devices, with 5-year validity.
|-
|Re-certification
|ICAR service for devices whose certification has expired after 5 years.
|-
|Competent Authority (CA)
|Ministry or organisation responsible for national animal identification schemes.
|-
|Voluntary sampling
|ICAR quality verification service for certified devices available on the market.
|}

== Scope ==
Section 10 of the ICAR Guidelines covers the testing and certification procedures, from the submission of the application by a manufacturer to the publication of the certification on the ICAR website, and the re-certification and/or sampling of the product.

Figure 1. Scope of Section 10: Testing and certification of animal identification devices
[[File:Imageoverview.png|center|thumb|582x582px|''Figure 1. Scope of Section 10: Testing and certification of animal identification devices'']]

== Application ==
The procedure for any type of test and certification starts with an application submitted by the manufacturer to the ICAR Secretariat. The Secretariat reviews the application, selects the test centre, issues an umbrella contract (for initial applicants), and issues the invoice which upon payment testing commences. Financial transactions between manufacturers, test centres and ICAR are coordinated by the ICAR Secretariat. Testing can only occur when the manufacturer sends all the necessary devices and accessories to the test centre. The devices and accessories remain the property of ICAR.

== Testing ==
Testing of identification devices can be subdivided into the following four main categories and outlined in Table 2.

=== RFID Conformance test (ISO 24631-1) ===
Conformance testing is required to demonstrate electronic transponders meet the specifications outlined in ISO 11784 and ISO 11785. The submission of identification devices to conformance testing is obligatory before they can be used in the official identification of animals.

Conformance tests are coordinated by the ICAR Secretariat. Acting as the RA on behalf of ISO, ICAR issues a Certificate for RFID devices conforming with ISO 11784 and ISO 11785.

Details of the RFID Conformance test are described in Procedure 1, Section 10 ‘Conformance of RFID Transponders with ISO Standards’ available in [https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Procedure_1:_Conformance_of_Transponders Procedure 1, Section 10 ‘Conformance of RFID Transponders with ISO 11784 and ISO 11785’].

=== RFID Performance test (ISO 24631-3) ===
Performance testing is an evaluation of the following characteristics of an RFID device: modulation amplitude, bit length stability, minimum activation field strength resonance frequency and amplitude voltage response (Vss). These RFID performance test results are not subject to pass or fail criteria but provide useful additional information on device behaviour when communicating with a reader. Acting as the RA on behalf of ISO, ICAR evaluates RFID devices through the RFID performance test and provides the report of the performance test to the manufacturer.

=== Device Composition and Environmental Performance test (ICAR) ===
ICAR offers a device composition and environmental performance test for both conventional and RFID external devices. The objective of these tests is to give extensive information on device durability and performance in diverse animal management environments. Procedures will vary depending on the device type. ICAR shares the test report and ICAR certificate with the manufacturer and the ICAR website is updated accordingly.

Details of the device composition and environmental performance test are described in [https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Procedure_4:_Testing_of_Conventional_Plastic_Ear_Tags Procedure 4, Section 10 ‘Testing of Conventional Plastic Ear Tags’] and [https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Procedure_5:_Testing_of_External_RFID_Devices Procedure 5, Section 10 ‘Testing of External RFID Devices’].

=== Voluntary sampling of Animal Identification Devices ===
Voluntary sampling is a service for Competent Authorities or other service users, other than manufacturers or their agents. The service is a quality verification service to ensure that devices available in the relevant market(s) remain compliant with the appropriate ISO and ICAR test protocols. Voluntary sampling does not lead to re-certification of the devices.

Details of the service are described in Procedure 6, Section 10 ‘Voluntary Sampling of Identification Devices’ are described in [https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Procedure_6:_Voluntary_sampling_of_Identification_Devices Procedure 6, Section 10 ‘Voluntary Sampling of Identification Devices’].

=== Summary of Tests ===
Table 2 summarizes the categories of tests.
<center>
{| class="wikitable"
| colspan="3" style="text-align:left;" |'''''Table 2. Categories for the testing of identification devices.'''''
|- style="background-color:#efefef;"
| style="text-align:left;" |'''Test category'''
| style="text-align:center;" |'''Test description'''
| style="text-align:center;" |'''Link to test procedure'''
|-
| style="text-align:left;" |Conformance and Performance ISO 24631-1
ISO 24631-1

ISO 24631-2

ISO 24631-3

ISO 24631-4
| style="text-align:center;" |Conformance/performance test of transponder (incl. granting of manufacturer code) or transceiver
| style="text-align:center;" |
* [https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Procedure_1:_Conformance_of_Transponders Procedure 1, Section 10] ‘Conformance of Transponders with ISO standards’

* [https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Procedure_2:_Granting_of_Manufacturer_Code Procedure 2, Section 10] ‘Granting of Manufacturer Code’

* [https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Procedure_3:_Conformance_of_Transceivers_with_ISO_Standards Procedure 3, Section 10] ‘Conformance of Transceivers with ISO standards’
|-
| style="text-align:left;" |Composition and environmental performance – Conventional ear tags
| style="text-align:center;" |Extended laboratory test
| style="text-align:center;" |
* [https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Procedure_4:_Testing_of_Conventional_Plastic_Ear_Tags Procedure 4, Section 10] ‘Testing of Conventional Plastic Ear Tags’
|-
| style="text-align:left;" |Composition and environmental performance – External RFID devices
| style="text-align:center;" |Extended laboratory test
| style="text-align:center;" |
* [https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Procedure_5:_Testing_of_External_RFID_Devices Procedure 5, Section 10] ‘Testing of External RFID Devices’
|-
| style="text-align:left;" |Voluntary sampling
| style="text-align:center;" |Partial test for certified devices available on the market.
| style="text-align:center;" |
* [https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Procedure_6:_Voluntary_sampling_of_Identification_Devices Procedure 6, Section 10] ‘Voluntary sampling of Identification Devices’
|}
</center>
=== Test Centres ===
Testing is conducted by ICAR-accredited test centres. Every test is contracted by the ICAR Secretariat to a specific test centre. The test centre is obliged to act according to the procedures laid down within the test protocols. In addition, all details associated with the testing phase, including the test results, must be kept strictly confidential.

Test centres attend the meetings of ICAR’s Animal Identification Sub-Committee and participate in annual ring test measurements to review consistency of test results between the laboratories. ICAR reviews the results to ensure overall uniformity between the laboratories. Further information regarding the test centres can be found [https://www.icar.org/index.php/technical-bodies/sub-committees/animal-identification-landing-page/animal-identification-sub-committee/ here].

== Manufacturer code ==
Following the first successful full conformance test, ICAR in its role as RA for ISO for the Standards 11784 and 11785 allocates to the manufacturer a code to be used only for products registered by ICAR. There are two types of manufacturer codes:

# Shared manufacturer code (900): can be granted to more than one manufacturer. A restricted range of identification codes is allocated to the registered product for exclusive use together with the shared manufacturer code.
# Unshared manufacturer code (901-998): can only be granted to one manufacturer following official proof that during two consecutive years the company has sold a minimum of one million (ICAR certified) transponders per year.

'''Note''': the manufacturer code concerns only the certification of RFID devices. With conventional ear tags, ICAR allocates unique certification codes to the products that pass the Device Composition and Environmental Performance Test.

== Report ==
Test centres prepare a confidential report of the test results and submit the report to the ICAR Secretariat. The Secretariat reviews the report and forwards it to the manufacturer along with the ICAR certificate should the test be successful. The report is also shared with the Animal Identification Sub-Committee for information only.

== Certification ==
The tests that lead to an ICAR certificate are:

# RFID Conformance test (ISO 24631-1).
# Device Composition and Environmental Performance test (ICAR).

Certificates are issued by the ICAR Secretariat and signed by the ICAR Chief Executive and then emailed to the manufacturer. In reference to certificates of conformance, the Chair of the ISO/TC23/SC19/WG3 is also copied in the communication to ensure the ISO remains informed about registered devices under the RA Agreement. For other tests not subject to pass or fail criteria (e.g. Performance test), an official ICAR letter acknowledging the completion of the test is sent to the manufacturer along with the test results.

== Publication ==
All ICAR-certified devices are published on the ICAR website:

# RFID devices web page (available [https://www.icar.org/index.php/rfid-devices-with-full-certification/ here]).
# Conventional ear tags web page (available [http://www.icar.org/index.php/certifications/animal-identification-certifications/conventional-ear-tags-for-bovine-and-ovine/ here]).

Devices whose certification has expired are removed from the above listed webpages. A specific web page (https://www.icar.org/index.php/rfid-iso/) lists all the devices registered by ICAR in conformance with ISO standards 11784 and 11785. Devices listed in this page are not removed as the registration is valid for the lifetime of the device.

''Table'' 3 summarizes the steps and responsibilities in the ICAR certification procedure.
<center>
{| class="wikitable"
| colspan="3" style="text-align:left;" |'''''Table 3. Steps, actions and responsibilities in the ICAR certification procedure'''''
|- style="background-color:#efefef;"
| style="text-align:left;" |'''Step'''
| style="text-align:center;" |'''Action'''
| style="text-align:center;" |'''Responsibility'''
|-
| style="text-align:left;" |1
| style="text-align:center;" |Application for testing of a device
| style="text-align:center;" |Manufacturer or dealer of identification device
|-
| style="text-align:left;" |2
| style="text-align:center;" |Acceptance of application, and issuance of umbrella contract and invoice
| style="text-align:center;" |ICAR Secretariat
|-
| style="text-align:left;" |3
| style="text-align:center;" |Testing and report compilation
| style="text-align:center;" |ICAR test centres
|-
| style="text-align:left;" |4
| style="text-align:center;" |Sharing of test results with the applicant
| style="text-align:center;" |ICAR Secretariat
|-
| style="text-align:left;" |5
| style="text-align:center;" |ICAR certification
| style="text-align:center;" |ICAR Secretariat
|-
| style="text-align:left;" |6
| style="text-align:center;" |Publication on the website
| style="text-align:center;" |ICAR Secretariat
|}
</center>
== Re-certification ==
After 5 years from the issuance of an ICAR certificate, the test can be repeated for the certification to be renewed for another 5 years. The device maintains its original product/certification code. The test protocols applied for the re-certification are:

# The limited test protocol for RFID devices
# The preliminary assessment protocol for conventional devices

'''Note''': if the application for re-certification is submitted more than 5 years after the original certification, full test procedures are required.

The application process is the same as for any other tests. Once re-certified, the device remains on the ICAR website for another 5 years and the updated expiration date of the certification is indicated..

== Voluntary sampling ==
At any given moment, Competent Authorities or other service users can apply for a sampling of certified devices found on the market. Devices are tested against the current ICAR standards, and the results are compared with original or re-certification results for the same devices. The test protocols used by the laboratories are:

# The limited test protocol for RFID devices.
# The preliminary assessment protocol for conventional devices.

The applicant may also request or specify additional test protocols, provided these are defined in other existing ISO or ICAR test protocols.

Devices to be tested must be collected and submitted to the ICAR test centre by the applicant and not by the manufacturer.

== Conditions for the use of ICAR certificates ==

# The conditions for the use of ICAR certificates are described in the respective procedures.
# If a device is certified by ICAR, the manufacturer may publish the certification of its device.
# IICAR certification does not guarantee that the device is suitable for all environments.
# If changes are made to a device during its 5-year certification period, the manufacturer must submit a Device Change Notification. See Procedures 4 and 5.

'''Note''': A manufacturer must not use the ICAR logo for any purpose, unless expressly authorised by ICAR.

== Appendices ==
Appendix A1. Application for RFID transponder Conformance test (ISO 24631-1) ([http://old.icar.org/wp-content/uploads/2016/03/Annex-A1-form-Section-10-Guidelines.pdf link])

Appendix A2. Application for a manufacturer code allocation ([http://old.icar.org/wp-content/uploads/2016/03/Annex-A2-form-Section-10-Guidelines.pdf link])

Appendix A3. Code of conduct ([https://www.icar.org/wp-content/uploads/documents/Annex-A3-Code-of-Conduct-2025.pdf link])

Appendix A4. Application for RFID transponder Performance test (ISO 24631-3) ([http://old.icar.org/wp-content/uploads/2016/03/Annex-A4-form-Section-10-Guidelines.pdf link])

Appendix A5. Application for RFID transceiver Conformance test (ISO 24631-2) ([http://old.icar.org/wp-content/uploads/2016/03/Annex-A5-form-Section-10-Guidelines.pdf link])

Appendix A6. Application for RFID transceiver Performance test (ISO 24631-4) ([http://old.icar.org/wp-content/uploads/2016/03/Annex-A6-form-Section-10-Guidelines.pdf link])

Appendix B1. Application for Device Composition and Environmental Performance test for conventional ear tags ([http://old.icar.org/wp-content/uploads/2017/04/Annex-B1-updated-March-2017.pdf link])

Appendix B2. Application for Device Change Notification (DCN) for conventional ear tags modified during the 5-year certification ([http://old.icar.org/wp-content/uploads/2017/04/Annex-B2-updated-March-2017.pdf link])

Appendix B3. Numbers for Reference Printing ([[Section 10 – Identification Device Certification#Appendix B3: Numbers of Reference Printing|link]]).

Appendix B4. Preliminary Test for Conventional Plastic Ear Tags ([[Section 10 – Identification Device Certification#Appendix B4: Preliminary Test for Conventional Plastic Ear Tags|link]]).

Appendix B5. Laboratory Test for Conventional Plastic Ear Tags ([[Section 10 – Identification Device Certification# Appendix B5: Laboratory Test for Conventional Plastic Ear Tags|link]]).

Appendix C1. Application for Device Composition and Environmental Performance test for external RFID devices ([http://old.icar.org/wp-content/uploads/2017/04/Annex-C1-updated-March-2017.pdf link])

Appendix C2. Application for Device Change Notification (DCN) for external RFID devices modified during the 5-year certification ([http://old.icar.org/wp-content/uploads/2017/04/Annex-C2-updated-March-2017.pdf link])

Appendix C3. Preliminary Test for External RFID Devices ([[Section 10 – Identification Device Certification#Appendix C3: Preliminary Test for External RFID Devices|link]]).

Appendix C4. Laboratory Test for External RFID Devices ([[Section 10 – Identification Device Certification#Appendix C4: Laboratory Test for External RFID Devices|link]]).

Appendix D1. Application for voluntary sampling of animal identification device ([https://old.icar.org/Guidelines/10-Appendix-D1-Application-for-voluntary-sampling-of-ICAR-certified-animal-identification-devices.pdf link])
[1] .
</div>
</div>

= Procedure 1: Conformance of Transponders =
<div class="mw-collapsible mw-collapsed">
<div>
== Foreword ==
ISO 24631 defines the test protocols for evaluating and verifying both the conformance (ISO 24631-1) and performance (ISO 24631-3) of RFID devices, and ISO 11784 defines the code structure. Only those results delivered by accredited and Registration Authority (RA) approved test centres are recognized.

== Introduction ==
ISO 11784 and ISO 11785 cover four RFID device types used for animal identification:

# Injectables: a small transponder able to be injected into an animal’s body and encapsulated in a biocompatible material with porosity equivalent to that of glass.
# Ear tag: a plastic covered transponder able to be fixed to an animal's ear using a locking mechanism which prevents the device from being removed without damaging it and rendering it unusable.
# Ruminal bolus: a transponder placed into a high specific gravity container orally administered to a ruminant animal where the device remains in the rumen of the animal due its high specific gravity which prevents its passing through the animal's digestive system.
# Tag attachment: a transponder covered by a primary protection layer but without its own locking mechanism and is used only as an attachment to a visual ear tag or to another means of external animal identification, e.g. leg tag, collar, etc.

The tests managed by ICAR as RA are recognised by the Federation of European Companion Animals Veterinary Association (FECAVA) and WSAVA (World Small Animal Veterinarian Association) and as such can be applied to companion animals also.

The fee for all tests will be borne by the applicant..

== References ==

* ISO 11784. Agricultural equipment - Radio frequency identification of animals - Code structure
* ISO 11785. Agricultural equipment - Radio frequency identification of animals - Technical concept
* ISO 24631-1. Agricultural equipment - Radio frequency identification of animals - Part 1: Evaluation of conformance of RFID transponders with ISO 11784 and ISO 11785 (including granting and use of a manufacturer code)
* ISO 24631-3. Agricultural equipment - Radio frequency identification of animals - Part 3: Evaluation of performance of RFID transponders conforming with ISO 11784 and ISO 11785
* ISO 3166. Codes for the representation of names of countries and their subdivisions

The latest version of ISO Standards will always apply and these Standards can be downloaded from the ISO website (https://www.iso.org/store.html).

== Procedures for verifying the ISO conformance of transponders ==

=== Application ===
A manufacturer can apply for:

# A full test; or
# A limited test; or
# A listing update.

'''A. A full test is mandatory in the following cases:'''

# When a non-RA registered manufacturer applies for a test.
# When a RA registered manufacturer uses a new silicon chip (Integrated Circuit) or implements new technology (HDX or FDX-B) in the transponder;
# When a RA registered manufacturer changes the coil technology (ferrite coils vs. air coils).

'''B. A limited test is applicable in the following cases:'''

# When a RA registered manufacturer inserts an ICAR certified transponder into a different primary transponder package.
# When a RA registered manufacturer uses the silicon chip of an ICAR certified transponder with different coil dimensions.
# When a RA registered manufacturer inserts an ICAR certified transponder with its original primary packaging into a different secondary packaging, e.g. a glass transponder into a bolus or a glass transponder into an ear tag.

'''C. A listing update is applicable in the following case:'''

# When a RA registered manufacturer intends to use an ICAR certified transponder without any modification. In this case the applicant must deliver a copy of the original test report along with a written confirmation from the ICAR registered manufacturer who originally submitted the transponder under question for certification by ICAR.

To apply for an ISO transponder conformance test, the manufacturer has to complete the test application form given in Appendix A1 (Application for RFID transponder Conformance test (ISO 24631-1)) which is available [http://old.icar.org/wp-content/uploads/2016/03/Annex-A1-form-Section-10-Guidelines.pdf here].

The completed application must be emailed in PDF format to the ICAR secretariat at [mailto:manufacturers@icar.org manufacturers@icar.org].

The manufacturer may choose their preferred ICAR accredited test centre. The manufacturer is required to submit to the test centre:

# 50 transponders for a full test, or
# 10 transponders for a limited test, or
# 10 transponders for a listing update.

The submitted transponders must have the ICAR test code of 999 or the existing manufacturer's code for a full test. The manufacturer can freely choose the transponder codes, but duplicate codes are not allowed. The manufacturer must provide a list of the transponder codes in decimal format.

Every device in a batch submitted for RFID testing (ISO 24631-1 and/or ISO 24631-3) must contain identical internal electronic components (coil and other components). Mixing of technologies (integrated circuit, capacitors, coils) within a single batch is prohibited. Likewise, when an electronic identification device is certified by ICAR, based on the results of testing, all devices that are released for sale must contain the same components as the original devices submitted for RFID testing. If changes are made to a device after approval, the device must be submitted for a new full or limited test, according to the type of changes as described above.

The test centre will test the transponders for compliance with ISO 11784 and ISO 11785. All tested transponders must be readable by the laboratory reference transceiver. The codes read by the laboratory reference transceiver must comply with ISO 11784 and the identification codes must be on the list of codes provided by the manufacturer.

The test centre will prepare a confidential report of the test results and will send the report to the ICAR secretariat.

The ICAR Secretariat will send the test report to the manufacturer and, in the case of a successful Conformance test result, an official ICAR letter of certification signed by the ICAR Chief Executive will also be sent to the manufacturer, with a copy to the ISO/TC23/SC19/WG3 Chair.

ICAR as RA issues a product code for each type of transponder successfully tested, including the listing update.

All electronic transponders submitted in an application will be kept by the test centre as reference transponders.

ICAR as RA maintains a public register on the ICAR website which lists all products registered and ICAR certified. A photograph of the certified device is included in the listing.

=== Conditions for the right to use an ICAR certificate for transponders (conformance test) ===
Upon successful completion of the Conformance test, ICAR will grant a device certificate valid for five years.

The ICAR certification of a transponder confirms the transponder's compliance with the code structure and the technical concepts given in ISO 11784 and ISO 11785.

The manufacturer must maintain a database register of all ICAR certified transponders sold. The manufacturer must require the initial purchasers of their ICAR certified transponders to also maintain a database register of their purchased product and require all subsequent purchasers to do the same until the transponder is applied to an animal.

The ICAR certificate is valid only for the transponder successfully tested and certified by ICAR. A manufacturer must not utilise the ICAR certificate for a transponder:

a. Which is not manufactured by them; and / or

b. Which does not comply in all respects with the ICAR certificate, including (but not limited to):

** Maintaining identical packaging (both primary and secondary) of the certified transponder.
** Maintaining identical technology and manufacturer of the certified transponder.
** Maintaining the identical transponder to the certified transponder.

c. Which utilises the manufacturer code of another manufacturer;

d. Which is supplied to or intended to be supplied to a person ("the receiver") who will market the transponder as if manufactured by them, unless:

** The receiver has obtained ICAR registration under this process; and
** The transponder bears either the shared manufacturer code or the unshared manufacturer code of the receiver.

Once the ICAR certification has been granted, the manufacturer will be responsible to:

# Keep an accurate and detailed log of all changes to their product and this log must be available to ICAR upon request. This log must include details of in-house performance measurements and Quality Assurance testing showing the amended product has maintained or enhanced its quality and performance.
# Submit the product for re-certification before the expiration of its current ICAR certification. The manufacturer must apply for re-certification not earlier than 6 months before the expiration of the certificate and no later than 5 months after the expiration of the certificate.
# Understand that ICAR may take sample products from the marketplace and test its conformance against the conformance of the device the manufacturer originally submitted, should ICAR suspect a breach of the signed ICAR Code of Conduct or a product change that has not been subjected to the tests outlined in this document - Procedure 1, Section 10 ‘Conformance of Transponders with ISO standards’.

Should the manufacturer fail to meet any or all of the above conditions for the use of the ICAR certificate, actions may be taken by ICAR in its role as Registration Authority for ISO according to the ISO standard 24631-1.

In cases of disputes regarding the conditions listed above or the use of an ICAR certificate, the decision of ICAR as RA will be binding.

ICAR as RA will distribute an advice notice regarding any manufacturer that distributes transponders in conflict with the certification procedure.
</div>
</div>

= Procedure 2: Granting of Manufacturer Code =
<div class="mw-collapsible mw-collapsed">
<div>
== Introduction ==
When countries have not implemented a procedure for the allocation and registration of the national identification code, a manufacturer code must be used instead of a country code to ensure a worldwide uniqueness of identification codes. ISO has appointed ICAR as the Registration Authority (RA) to allocate manufacturer codes in conformance with ISO 11784.

== Application of shared and unshared manufacturer code ==

=== Shared manufacturer code ===
A manufacturer code can be granted to more than one manufacturer, and this code is known as a shared manufacturer code. A shared manufacturer code is granted by ICAR if the manufacturer's RFID device has successfully completed a full conformance test. When a shared manufacturer code is granted, ICAR also allocates a restricted set of identification codes for exclusive use with the shared manufacturer code. The identification codes allocated in combination with the shared manufacturer code are unique. ICAR must ensure this uniqueness by applying appropriate procedures for the assignment and registration of allocated identification codes. If necessary, additional sets of identification codes can be assigned to the manufacturer by request. The size of the sets of allocated identification codes is determined on consensual agreement with the manufacturer and ICAR..

=== Unshared manufacturer code ===
An unshared manufacturer code will only be granted to a manufacturer providing proof to the Registration Authority that during two consecutive years the company has sold a minimum of one million (ICAR certified) transponders per year. This proof must be sourced from their sales records and certified by their external auditor of accounts or a notary public.

== Manufacturer code application procedure ==
The first time a manufacturer applies for a conformance test, the manufacturer must also apply for a manufacturer code and sign a Code of Conduct.

The intial application must consist of a completed test application form ([http://old.icar.org/wp-content/uploads/2016/03/Annex-A1-form-Section-10-Guidelines.pdf Appendix A1]), a completed application form for a manufacturer code ([http://old.icar.org/wp-content/uploads/2016/03/Annex-A2-form-Section-10-Guidelines.pdf Appendix A2]), and the signed "Code of Conduct" ([https://www.icar.org/wp-content/uploads/documents/Annex-A3-Code-of-Conduct-2025.pdf Appendix A3]). By signing the application form and the Code of Conduct, the manufacturer agrees to fulfil the conditions described in this document and to accept the charges for this procedure.

The completed application must be emailed to the ICAR secretariat at: [mailto:manufacturers@icar.org manufacturers@icar.org] [[Mailto:manufacturers@icar.org|manufacturers@icar.org]].

ICAR maintains a public register listing all registered products, manufacturers, and manufacturer codes.

== Conditions for the right to use the manufacturer code ==
The manufacturer must only use their manufacturer code for products registered by ICAR.

In disputes regarding the conditions of manufacturer code use, the decision of ISO/TC23/SC19 will be binding.

For further reference, ISO 11784 can be downloaded from the ISO web site (https://www.iso.org/store.html).

== The use of Manufacturer codes and Country codes ==

In accordance with ISO 11784 and Section 10 of these Guidelines, including [https://www.icar.org/wp-content/uploads/documents/Annex-A3-Code-of-Conduct-2025.pdf Appendix A3] Code of Conduct, manufacturer codes (900-998 series) should only be used in connection with electronic identification (RFID) devices.

Where a competent national authority has assumed the responsibility for ensuring and maintaining the uniqueness of the RFID identification code for a specific species in that country, the ISO 3166 3-digit numeric country code may be used in place of the manufacturer code in the electronic identification of that specific species of animal. A country code may only be used if it is authorized by the competent authority of that country.

The use of manufacturer codes in the International Identity used for genetic evaluation purposes is discouraged ([[Section 09 – Dairy Cattle Genetic Evaluation|Section 9 – Dairy Genetic Evaluations]]).

== The use of manufacturer codes and country codes ==
In accordance with ISO 11784 and Section 10 of these Guidelines, including [https://www.icar.org/wp-content/uploads/documents/Annex-A3-Code-of-Conduct-2025.pdf Appendix A3] Code of Conduct, manufacturer codes (900-998 series) should only be used in connection with electronic identification (RFID) devices.

Where a competent national authority has assumed the responsibility for ensuring and maintaining the uniqueness of the RFID identification code for a specific species in that country, the ISO 3166 3-digit numeric country code may be used in place of the manufacturer code in the electronic identification of that specific species of animal. A country code may only be used if it is authorized by the competent authority of that country.

The use of manufacturer codes in the International Identity used for genetic evaluation purposes is discouraged ([[Section 09 – Dairy Cattle Genetic Evaluation|Section 9 – Dairy Genetic Evaluations]]).
</div>
</div>

= Procedure 3: Conformance of Transceivers with ISO Standards =
<div class="mw-collapsible mw-collapsed">
<div>
== Scope ==
This section refers to ISO 24631-2 and ISO 24631-4, regarding the test procedures to verify the compliance of RFID transceivers to the operating characteristics outlined in ISO 11784 and ISO 11785.

== References ==
The titles of standards referred to in this document are as follows:

* ISO 11784 Radio frequency identification of animals - Code structure
* ISO 11785 Radio frequency identification of animals - Technical concept
* ISO 24631-2 Radiofrequency identification of animals - Part 2: Evaluation of conformance of RFID transceivers with ISO 11784 and ISO 11785
* ISO 24631-4 Radiofrequency identification of animals - Part 4: Evaluation of performance of RFID transceivers conforming with ISO 11784 and ISO 11785
* ISO 3166 Codes for the representation of names of countries and their subdivisions

The latest version of ISO Standards will always apply, and these Standards are available on the ISO website (https://www.iso.org/store.html).

== Manufacturer application ==
</div>Manufacturers seeking a conformance test of a tranceiver must complete the test application form ([http://old.icar.org/wp-content/uploads/2016/03/Annex-A5-form-Section-10-Guidelines.pdf Appendix A5]), and email it to the ICAR secretariat at [mailto:manufacturers@icar.org manufacturers@icar.or]

== ICAR testing ==
Upon receipt of the application, the ICAR Secretariat will assign one of the accredited test centres to carry out the conformance test on the nominated transceiver. The manufacturer’s approved test centre may be taken into consideration.

The transceiver will be tested against the ISO procedures listed in the Scope of this procedure to verify compliance to the operationing characteristics outlined in ISO 11784 and and ISO 11785.

== Conclusion of the laboratory and certification tests ==
The test centre will prepare a test report and will submit it to the ICAR Secretariat. All information collected during the laboratory tests will remain confidential and only disclosed to the manufacturer of the transceiver.

Upon the successful completion of the laboratory test, ICAR will send the test report and an official letter to the manufacturer granting ICAR certification for that transceiver. Certified tranceivers are listed on the ICAR website inclusive of a photograph of the transceiver.

If the laboratory test results are unsatisfactory, ICAR will send the manufacturer the test report indicating the reasons for the failure.

All test reports are shared with ICAR’s Animal Identification Sub-Committee for information.
</div>

= Procedure 4: Testing of Conventional Plastic Ear Tags =
<div class="mw-collapsible mw-collapsed">
<div>
== Introduction ==
This section will guide the manufacturer through the steps of obtaining and retaining ICAR certification for a conventional permanent plastic ear tag.

The ICAR procedures for testing the performance and reliability of permanent identification devices consider, but are not limited, to the following issues:
# Ease of application and use.
# Efficiency of animal recognition.
# Durability and single-use design characteristics.
# Animal welfare and human health.

The following procedures focus on testing the ear tag design, the print quality and, if requested, the ear tag machine readability.

The testing procedure is composed of three distinct phases:

# Phase 1: Manufacturer's application (section 5.1).
# Phase 2: Preliminary Assessment (section 5.2).
# Phase 3: Laboratory Test (section 5.4).

These test procedures must be carried out by an ICAR accredited test laboratory. The fees for these test procedures will be borne by the device manufacturer.

When an ear tag is certified by ICAR, the manufacturer will be authorized to state that tags of that particular design and printing method are ICAR certified. ICAR certification does not imply that the tag is suitable for all environments or that its machine-readable characteristics are satisfactory for all uses. It is the manufacturer's responsibility to comply with the requirements of the relevant jurisdictions.

A successfully tested and certified product can have its certification withdrawn if the product fails to comply with the requirements described in this section. ICAR and/or national authorities may randomly take samples of certified tags from the market and subject them to testing to ensure certified ear tags continue to meet ICAR certification criteria. The manufacturer will be required to meet the costs of these assessments should the product fail to meet ICAR standards.

The manufacturer must advise ICAR of any changes made affecting the performance of ICAR certified products which could alter their previous test results. The manufacturer must also inform ICAR of any change to the material composition or the print quality of a certified ear tag.

Users of ear tags and / or potential users of ear tags are encouraged to access the list of certified tags found on the ICAR web site (page available [http://www.icar.org/index.php/certifications/animal-identification-certifications/conventional-ear-tags-for-bovine-and-ovine here]).

== Scope ==
This section describes the procedures for measuring the composition and the performance of conventional permanent plastic ear tags which may include machine readable printing.

When a manufacturer submits an ear tag to ICAR for testing, they may also choose to have the machine readability of the ear tag evaluated according to this protocol. If such request is not explicitely made, then only the visual readability will be evaluated.

Successful completion of the procedures described in this section will result in the ICAR certification of the ear tag as a device recommended by ICAR for animal identification purposes. ICAR certified ear tags are published on the ICAR website here.

Figure 1 gives a summary of the main elements of the testing and certification process of conventional ear tags.

[[File:Imagetestn.png|center|thumb|491x491px|''Figure 2. Key steps for the testing and certification of conventional ear tags'' ]]

== References ==
{| class="wikitable"
| colspan="2" |'''''Table 4. References to relevant standards.'''''
|-
|ISO 175
|Resistance of thermoplastics to liquids
|-
|EN 1122
|Plastics - Determination of cadmium - Wet decomposition method
|-
|ISO 1817
|Resistance of vulcanized elastomers to liquids
|-
|ISO 4650
|Rubber - Identification - Infrared spectrometric method
|-
|ISO 9924
|Determination of composition of vulcanized elastomers
|-
|ISO 11357
|Plastics - Differential scanning calorimetry (DSC)
|-
|ISO 9352
|Plastics - Determination of resistance to wear by abrasive wheels
|-
|ISO 527-1
|Plastics - Determination of tensile properties part 1: General principles
|-
|ISO 37
|Rubber, vulcanized or thermoplastic - Determination of tensile stress-strain properties
|-
|ISO 4611
|Plastics - Determination of the effects of exposure to damp heat, water spray and salt mist
|-
|EN ISO 4892-2
|Plastics - Methods of exposure to laboratory light sources - Part 2: Xenon-arc lamps
|-
|ISO 15416
|Information technology - Automatic identification and data capture techniques - Bar code print quality test specification; Linear symbols
|-
|ISO 11664-4
|Colorimetry - Part 4: CIE 1976 L*a*b* Colour space
|-
|ISO 105-X12
|Textiles – Tests for colour fastness – Part X12: Colour fastness to rubbing
|}

The latest version of the above references will always apply.

== Definitions ==

=== Certification code ===
A certification code is an alpha-numeric code consisting of "A", followed by three numbers. The certification code is used to identify and register an ear tag model that has successfully passed the testing procedure. This code may be embossed on all ICAR certified ear tags for official identification. The placement of the certification code on the ear tag should conform to the relevant jurisdictional requirements in whatever locality the ear tag is sold.

=== Certified ear tag ===
A certified ear tag is an ear tag that was submitted to the ICAR accredited test centre where it successfully passed the testing procedures and was thus certified by ICAR.

=== Ear tag ===
An ear tag is deemed to be composed of three principal features:

# The front plate which is often, but not always, the "female" component of an ear tag combination. The front plate is designated as such because it will be in the front of the animal's ear when the ear tag combination is applied correctly.
# The rear plate which is often, but not always, the "male" component of an ear tag combination. The rear plate is designated as such because it will be at the back of the animal's ear when the ear tag combination is applied correctly.
# The locking mechanism which comprises of the locking gap in the female component of an ear tag and the pin of the male component of the ear tag combination.

=== Manufacturer ===
The manufacturer is the company or person submitting the application for the testing of an ear tag and has accepted the ICAR conditions for certification of conventional permanent plastic ear tags as outlined in section 5.4.6.

=== Reference colour ===
The colour of the ear tags used in the laboratory tests must be yellow and the colour of the printing must be black. The manufacturer must print a uniform solid block 10mm x 10mm in the same colour as the colour of the printing on the tag.

=== Reference number ===
Printing must be composed of four different and predefined figures (from 0 to 9) as outlined in [http://www.icar.org/Guidelines/10-Appendix-B3-Numbers-for-Reference-Printing.pdf Appendix B3]. The font style and size must replicate precisely the font style and size the manufacturer commonly uses on that tag within the market.

For the ear tags where machine readability will be assessed, a 12-digit barcode must be printed on the tags in addition to the reference number. The 12-digit barcode consists of the three numbers of the test code as defined in section 4.7 followed by zeroes and the reference number.

=== Test code ===
The test code is an alpha-numeric code consisting of "T" (for ‘tested’), followed by 3 numbers. The test code is allocated by ICAR upon the completion of a successful Preliminary assessment.

The test code is used to identify and register an ear tag model being tested at the laboratory under the approval procedure. This code must be printed or engraved on all ear tags undergoing testing during the approval procedure.

=== Tested ear tag ===
A tested ear tag is an ear tag that was submitted to the ICAR accredited test centre and subsequently tested.

== ICAR testing and certification procedure ==

=== Phase 1: Manufacturer's application ===
To submit an ear tag for ICAR testing within the scope of the tests described in this section, the manufacturer must complete an application and email it in PDF format to the ICAR secretariat at manufacturers@icar.org

The application must consist of:

# The Application Form ([http://www.icar.org/wp-content/uploads/2017/04/Annex-B1-updated-March-2017.pdf Appendix B1] or [http://old.icar.org/wp-content/uploads/2016/03/Annex-A2-form-Section-10-Guidelines.pdf Appendix B2]):
** Appendix B1 is the application form for the certification of a new device or re-certification of an already certified device.
** Appendix B2 is the application form for the certification of a device modified during its certification. (Please refer to section 6 for information on the Device Change Notification)

Copies of the required application forms can be obtained from the ICAR website or from the ICAR Secretariat.

When a manufacturer chooses to have the machine readable printing on the ear tag evaluated, the manufacturer must indicate this choice on the completed Application Form. The application should also specify the symbols (language) used on the tag, e.g. Quick Response (QR) Model 2, Data Matrix (DM) ECC 200, Aztec, Code 128, Code 39 or Interleaved 2 of 5. The applicant should also indicate if the AIM (Automatic Identification Manufacturers International Inc) quality standards (code dimensions) have been met.

By signing the application form, the manufacturer agrees to fulfil the conditions of ICAR testing, certification and payment obligations and also acknowledges the ongoing monitoring and assessments for certified ear tags.

=== Phase 2: Preliminary assessment ===
To assess conformance of the ear tags with the information given in the application form and to also detect any major failure, e.g. damage of the tag at application, possible unlocking without deformation, inappropriate animal welfare design etc., the ear tags will be submitted to a Preliminary Assessment.

The Preliminary Assessment procedure is also applied to a device for which the manufacturer is requesting re-certification.

Refer to [https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Appendix_B4:_Preliminary_Test_for_Conventional_Plastic_Ear_Tags Appendix B4] for details.

== Conclusion of the Preliminary assessment ==
The test centre will prepare a comprehensive report detailing the results of the Preliminary Assessment. This report will be submitted to the ICAR Secretariat, who will then forward the test report to the manufacturer along with detailed instructions for the submission of tags for the laboratory test. The Preliminary Assessment will expire six months after a successful result. Tags may not be submitted for a laboratory test after the Preliminary Assessment has expired and will need to be re-submitted for another Preliminary Assessment.

If a device has not performed satisfactorily, ICAR will provide the manufacturer with the test report and indicate the reasons for the tag's failure.

== Phase 3: Laboratory Test ==

=== Assigning a test centre ===
Following the successful completion of the Preliminary Assessment, the ICAR Secretariat will assign one of the accredited test centres to carry out the Laboratory Test. The manufacturer’s preferred approved test centre may be taken into consideration.

=== Granting of a test code ===
A specific test code will be allocated by ICAR for the ear tag undergoing testing. See Section 4.7 above.

=== Manufacturer requirements ===
At the commencement of the Laboratory Test, the manufacturer must deliver the following items to the assigned test centre:

# 200 yellow ear tags with the test code number and the reference printing applied (including the uniform solid block described in 4.5). For tags where the machine readability is to be assessed, a 12-digit barcode must also be printed on the ear tag. Note: the manufacturer will be allocated 25 reference numbers to print on the 200 ear tags, i.e. 8 tags per reference number ([http://www.icar.org/Guidelines/10-Appendix-B3-Numbers-for-Reference-Printing.pdf Appendix B3]).
# One tag applicator or an equivalent device supplied for the application of devices to animals.
# A statement specifying the nature of the polymer used for the device.
# A statement advising if any regrind material has been used in any component of the device.

=== Test procedure ===
The test procedure to be followed for Phase 3: Laboratory Test is described in [https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Appendix_B5:_Laboratory_Test_for_Conventional_Plastic_Ear_Tags Appendix B5]'''.'''

=== Conclusion of the laboratory test ===
The test centre will prepare a test report and will submit it to the ICAR Secretariat. All information collected during the laboratory tests will remain confidential and only disclosed to the manufacturer of the ear tag.

Upon the successful completion of the Laboratory Test, ICAR will send the test report and an official letter to the manufacturer granting ICAR certification for that ear tag.

If the manufacturer had requested an evaluation on the machine readability of the ear tag, then this evaluation will also be included in the test report.

Each test report on a successfully tested ear tag will include a summary sheet with an evaluation of the appropriate suitability of the ear tag for various production systems and / or environmental conditions.

If the Laboratory Test results are unsatisfactory, ICAR will send the manufacturer the test report indicating the reasons for the failure.

All test reports are shared with ICAR’s Animal Identification Sub-Committee for information.

== ICAR conditions for certification of conventional permanent plastic ear tags ==

# Upon successful completion of the ICAR test procedures described above, ICAR will grant a device certificate valid for five years and a certification code.
# This certification is valid only for the specific plastic ear tag type successfully tested and certified by ICAR.
# A manufacturer cannot utilise the ICAR certification for a plastic ear tag:
#* Which is not manufactured by them; or
#* Which does not comply in all respects to the ICAR certification, which includes maintaining an identical tag type to the certified tag.
# Once the ICAR certificate has been granted, the manufacturer will be responsible to:
#* Keep an accurate and detailed log of all changes to their product and this log must be available to ICAR upon request. This log must include details of in-house performance measurements and Quality Assurance testing showing the product has maintained or enhanced its quality, performance and material composition.
#* Submit the product for a Device Change Notification (DCN – [http://old.icar.org/wp-content/uploads/2016/03/Annex-A2-form-Section-10-Guidelines.pdf Appendix B2]) if changes are made to the device during its 5-year certification period. At the determination of the ICAR Secretariat, the modified device may require a new certification code and the manufacturer will need to declare if the modified device will replace the existing device or if the two devices are going to co-exist. Every DCN application will be reviewed individually by ICAR and the designated laboratory, and ICAR shall decide if a limited test is applicable, or if the significance of the modification(s) requires a full test. '''''Note''': The request for DCN is not applicable to all types of changes to a device. Manufacturers are requested to contact the ICAR Secretariat for guidance before they apply for DCN by emailing to: [mailto:manufacturers@icar.org manufacturers@icar.org].''
#*Submit the product for re-certification before the expiration of its current ICAR certification. The manufacturer must apply for re-certification no earlier than 6 months before the expiration of the certificate and no later than 5 months after the expiration of the certificate.
#*Understand that within the 5 year timeframe, should ICAR suspect a product change that has not been subjected to the tests outlined in this Procedure 4 of Section 10 of the ICAR Guidelines, or any other breach of the conditions described in this chapter, ICAR may take sample products from the market and test their performance against the performance of the certified device.
# Should the manufacturer fail to meet any or all the above certification conditions, ICAR may withdraw the certification.
# In disputes regarding the conditions above or the use of a certificate, the decision of ICAR will be binding.
# ICAR will distribute an advice notice regarding any manufacturer distributing products in conflict with the testing and certification procedures outlined in this Procedure 4 of Section 10 of the ICAR Guidelines.
</div>
</div>

= Procedure 5: Testing of External RFID Devices =
<div class="mw-collapsible mw-collapsed">
<div>
== Introduction ==
This section will guide the manufacturer through the steps of obtaining and retaining ICAR certification for an external permanent radio frequency identification (RFID) device.

The ICAR procedures for testing the performance and reliability of external permanent RFID devices consider, but are not limited to the following issues:
# Ease of application and use.
# Efficiency of animal recognition.
# Durability and tamper-evidence.
# Animal welfare and human health.

Only external RFID devices designed as permanent electronic identification devices are covered in this procedure 5 of Section 10 of the ICAR Guidelines.

The testing procedure is composed of three distinct phases:
# Phase 1: Manufacturer’s application ([https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Phase_1_Manufacturer%E2%80%99s_Application section 6.5.1] )
# Phase 2: Preliminary assessment ([https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Phase_2:_Preliminary_Assessment_2 section 6.5.2])
# Phase 3: laboratory Test ([https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Phase_3_Laboratory_Test section 6.5.3])

These test procedures must be carried out by an ICAR accredited test laboratory. The fees for these test procedures will be borne by the device manufacturer.

When an ear tag is certified by ICAR, the manufacturer will be authorized to state that tags of that particular design and printing method are ICAR certified. ICAR certification does not imply that the tag is suitable for all environments or that its machine-readable characteristics are satisfactory for all uses. It is the manufacturer's responsibility to comply with the requirements of the relevant jurisdictions.

A successfully tested and certified product can have its certificate withdrawn if the product fails to comply with the requirements described in this section. ICAR and/or national authorities may randomly take samples of certified devices from the market and subject them to testing to ensure certified devices continue to meet ICAR certification criteria. The manufacturer will be required to meet the costs of these assessments should the product fail to meet ICAR standards.

The manufacturer must advise ICAR of any changes made affecting the performance of ICAR certified products which could alter their previous test results. The manufacturer must also inform ICAR of any change to the material composition of a certified RFID device.

Users (and/or potential users) of external RFID devices are encouraged to access the list of certified RFID devices found on the ICAR web site [https://www.icar.org/index.php/rfid-devices-with-full-certification/ here].

== Scope ==
This section describes the procedures for measuring the composition and the performance of external RFID devices.

Successful completion of the procedures described in this section will result in the ICAR certification of the tested RFID device as a device recommended by ICAR for animal identification purposes. ICAR certified RFID devices are published on the ICAR web site ([https://www.icar.org/index.php/rfid-devices-with-full-certification/ here]).

Figure 3 gives a summary of the main elements of the testing and certification process of external RFID devices.
[[File:Imagefig3.png|center|thumb|457x457px|''Figure 3. Key steps for the testing and certification of external RFID devices'']]

== References ==
<div align="center">
<table class="MsoNormalTable" border="0" cellspacing="0" cellpadding="0" style="border-collapse: collapse" id="table1">
<tr style="page-break-inside: avoid">
<td valign="top" style="width: 613px; border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" colspan="2">
''Table 3. References to relevant standards''
</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width: 118.8pt; border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">

EN 1122</td>
<td width="455" valign="top" style="width: 341.4pt; border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">

Plastics - Determination of cadmium - Wet decomposition method</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

ISO 4650</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Rubber - Identification - Infrared spectrometric method</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

ISO 9924</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Determination of composition of vulcanized elastomers</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

ISO 11357</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Plastics - Differential scanning calorimetry (DSC)</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

ISO 527-1</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Plastics - Determination of tensile properties part 1: General principles</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

ISO 37</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Rubber, vulcanized or thermoplastic - Determination of tensile stress-strain properties</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

ISO 11664-4</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Colorimetry - Part 4: CIE 1976 L*a*b* Colour space</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

ISO 7724

ISO 105-X12</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Paints and Varnishes – Colorimetry

Textiles – Tests for colour fastness – Part X12: Colour fastness to rubbing</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

EN ISO 4892-2</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Plastics - Methods of exposure to laboratory light sources Part 2: Xenon-arc lamps</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

EN/IEC 60068-2-1</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Environmental testing - Part 2-1: Tests - Test A: Cold</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

EN/IEC 60068-2-2</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Environmental testing - Part 2-2: Tests - Test B: Dry heat</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

EN/IEC 60068-2-32</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Environmental testing - Part 2-32: Tests - Test Ed: Free fall</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

ISO 4611</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Plastics - Determination of the effects of exposure to damp heat, water spray and salt mist</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

ISO 11785</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Radio frequency identification of animals - Technical concept</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width:118.8pt;padding:0cm 5.4pt 0cm 5.4pt">

ISO 24631-1</td>
<td width="455" valign="top" style="width:341.4pt;padding:0cm 5.4pt 0cm 5.4pt">

Radio frequency identification of animals - Part 1: Evaluation of conformance of RFID transponders with ISO 11784 and ISO 11785</td>
</tr>
<tr style="page-break-inside: avoid">
<td width="158" valign="top" style="width: 118.8pt; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">

ISO 24631-3</td>
<td width="455" valign="top" style="width: 341.4pt; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">

Radio frequency identification of animals - Part 3: Evaluation of performance of RFID transponders conforming with ISO 11784 and ISO 11785</td>
</tr>
</table>
</div>

The latest version of the above references will always apply.
== Definitions ==

=== Certification code ===
A certification code is an alpha-numeric code consisting of “A”, followed by three numbers. The certification code is used to identify and register an RFID device that has successfully completed the testing procedure. This code may be embossed or printed on all ICAR certified RFID devices for official identification. The placement of the certification code on the device should conform to the relevant jurisdictional requirements in whatever the locality the RFID device is sold.

=== Certified RFID device ===
A certified RFID device is an RFID device that was submitted to the ICAR accredited test centre where it successfully passed the testing procedures and was thus certified by ICAR.

=== Manufacturer ===
The manufacturer is the company or person submitting the application for the testing of an RFID device and has accepted the conditions of ICAR for the certification of external RFID devices as outlined in section 7.

=== Reference colour ===
The colour of the external RFID device used in the laboratory tests must be yellow and the colour of the printing must be black. On the test samples, preferably on the rear part, the manufacturer must print a uniform solid block of 10mm x 10mm in the same colour as the colour of the printing on the device. Should the surface area of the device be too small to accommodate the printing of a 10mm x 10mm solid block, then a uniform solid block of 5mm x 20mm is acceptable. This printing may be on the female tag plate or on the male tag plate (sometimes known as the pin).

=== Test code ===
The test code is an alpha-numeric code consisting of "T" (for ‘tested’), followed by 3 numbers. The test code is allocated by ICAR upon the completion of a successful Preliminary Assessment.

The test code is used to identify and register an ear tag model being tested at the laboratory under the approval procedure. This code must be printed or engraved on all ear tags undergoing testing during the approval procedure.

=== Reference ID codes ===
The transponders of the RFID devices submitted to the laboratory test must be programmed with the test code of 999 followed by zeroes and a sequential numerical code as per the following:

# For the Phase 2 Preliminary Assessment, the sequential numerical code range will be: 001 - 130.
# For the Phase 3 Laboratory Test, the sequential numerical code range will be: 201 - 400.
# The reference ID code programmed into each transponder must be printed on the front part of each device. The font style and size must replicate precisely the font style and size the manufacturer commonly uses on that device within the market. This font size and style must be specified in the application form ([http://www.icar.org/wp-content/uploads/2017/04/Annex-C1-updated-March-2017.pdf Appendix C1] or [http://www.icar.org/wp-content/uploads/2017/04/Annex-C2-updated-March-2017.pdf Appendix C2]).

=== RFID ear tag ===
An RFID ear tag is a radio frequency identification (RFID) external device able to be fixed to an animal’s ear and deemed to be composed of three principal features:

# The front part which is often, but not always, the “female” component of an ear tag combination. The front part is designated as such because it will be in the front of the animal’s ear when the ear tag combination is applied correctly. It will often, but not always, contain the transponder.
# The rear plate which is often, but not always, the “male” component of an ear tag combination. The rear plate is designated as such because it will be at the back of the animal’s ear when the ear tag combination is applied correctly.
# The locking mechanism which comprises of the locking gap in the female component of an ear tag and the pin of the male component of an ear tag combination

=== RFID leg tag ===
An RFID leg tag is a radio frequency identification (RFID) external device able to be permanently fastened to an animal’s lower leg.

=== Tested RFID device ===
A tested RFID device is a device that was submitted to the ICAR approved test centre and subsequently tested

== ICAR Testing and Certification Procedure ==

=== Phase 1 Manufacturer’s Application ===
To submit an external RFID device for ICAR testing within the scope of the tests described in this section, the manufacturer must complete an application and email it in PDF format to the ICAR secretariat at [mailto:Manufacturers@icar.org manufacturers@icar.org]

The application must consist of:
# The Application Form ([http://www.icar.org/wp-content/uploads/2017/04/Annex-C1-updated-March-2017.pdf Appendix C1] or [http://www.icar.org/wp-content/uploads/2017/04/Annex-C2-updated-March-2017.pdf Appendix C2]):
#* Appendix C1 form is the application form for the certification of a new device.
#* Appendix C2 is the application form for the certification of a device that has been modified during its certification period. (Please refer to section 7 for information on the Device Change Notification).
Copies of the required application form can be obtained from the ICAR web site ([http://www.icar.org/index.php/certifications/animal-identification-certifications/application-forms-for-testing-of-id-devices/ here]) or from the ICAR Secretariat.

By signing the application form, the manufacturer agrees to fulfil the conditions of ICAR testing, certification and payment obligations and also acknowledges the ongoing monitoring and assessments applicable for certified RFID devices.

=== Phase 2: Preliminary Assessment ===
To assess conformance of the ear tags with the information given in the application form and to also detect any major failure, e.g. damage of the tag at application, possible unlocking without deformation, inappropriate animal welfare design etc., the ear tags will be submitted to a Preliminary Assessment.

The Preliminary Assessment procedure is also applied to a device for which the manufacturer is requesting re-certification.

Refer to detailed test procedure in [[Section 10 – Identification Device Certification#Appendix C3: Preliminary Test for External RFID Devices|Appendix C3]] (Preliminary Test for External RFID Devices)

=== Conclusion of the Preliminary assessment ===
The test centre will prepare a comprehensive report detailing the results of the Preliminary Assessment. This report will be submitted to the ICAR Secretariat, who will then forward the test report to the manufacturer along with detailed instructions for the submission of tags for the laboratory test. The Preliminary Assessment will expire six months after a successful result. Tags may not be submitted for a laboratory test after the Preliminary Assessment has expired and will need to be re-submitted for another Preliminary Assessment.

If a device has not performed satisfactorily, ICAR will provide the manufacturer with the test report and indicate the reasons for the tag's failure.

=== Phase 3 Laboratory Test ===

==== Assigning a Test Centre ====
Following the successful completion of the Preliminary Assessment, the ICAR Secretariat will assign one of the accredited test centres to carry out the Laboratory Tests. The manufacturer’s preferred approved test centre may be taken into consideration.

==== Granting of a Test Code ====
A specific test code will be allocated by ICAR and this code must be printed or engraved on all ear tags undergoing testing. See Paragraph 4.5.

==== Manufacturer requirements ====
At the commencement of the Laboratory Test, the manufacturer must deliver the following items to the assigned test centre:

# 200 external RFID devices programmed with the reference ID codes. The test code and reference printing ([http://www.icar.org/Guidelines/10-Appendix-B3-Numbers-for-Reference-Printing.pdf Appendix B3]) must printed or engraved on the exterior of each device.
# One tag applicator or an equivalent device supplied for the application of devices to animals.
# A statement specifying the nature of the polymer used for the device.
# A statement advising if any regrind material has been used in any component of the device.

==== Test procedure ====
Refer to the detailed test procedures in [[Section 10 – Identification Device Certification#Appendix C4: Laboratory Test for External RFID Devices|Appendix C4]] (Laboratory Test for External RFID Devices)

== Conclusion of the laboratory tests ==
The test centre will prepare a test report and will submit it to the ICAR Secretariat. All information collected during the laboratory tests will remain confidential and only disclosed to the manufacturer of the RFID device.

Upon the successful completion of the laboratory test, ICAR will send the test report and an official letter to the manufacturer granting ICAR certification for that RFID device.

Each test report on a successfully tested RFID device will include a summary sheet with an evaluation of the appropriate suitability of the RFID device for various production systems and / or environmental conditions.

If the Laboratory Test results are unsatisfactory, ICAR will send the manufacturer a test report indicating the reasons for the failure.

All test reports are shared with ICAR’s Animal Identification Sub-Committee for information.

== ICAR conditions for certification of conventional permanent plastic ear tags ==

# Upon successful completion of the ICAR test procedures described above, ICAR will grant a device certificate valid for five years and a certification code .
# The certification is valid only for the specific external RFID device type successfully tested and certified by ICAR.
# Manufacturers can only utilise the ICAR certification of a device under the following conditions:
#* The device must be manufactured by them;
#* The device must comply in all respects to the original ICAR certification (maintaining identical technology and production process, the same RFID transponder, etc.). If any modifications are made to the certified device, refer to the following point.
# Once the ICAR certification has been granted, the manufacturer will be responsible to:
#* Keep an accurate and detailed log of all changes to their product and this log must be available to ICAR upon request. This log must include details of in-house performance measurements and Quality Assurance testing showing the product has maintained or enhanced its quality, performance and material composition.
#* Submit the product for a Device Change Notification (DCN – [https://www.icar.org/wp-content/uploads/2017/04/Annex-C2-updated-March-2017.pdf Appendix C2]) when changes are made to the composition and environmental performance characteristics of the device during its 5-year certification period. At the determination of the ICAR Secretariat,the modified device may require a new certification code, and the manufacturer will need to declare if the modified device will replace the existing device or if the two devices are going to co-exist. Every DCN application will be reviewed individually by ICAR and the designated laboratory, and ICAR shall decide if a limited test is applicable, or if the significance of the modification(s) requires a full test. '''''Note''': The request for a DCN is not applicable to all types of changes to a device. Manufacturers should contact the ICAR Secretariat ([mailto:manufacturers@icar.org manufacturers@icar.org]) for guidance before they apply for DCN.''
#*Submit the product for re-certification before the expiration of its current ICAR certification. The manufacturer must apply for re-certification no earlier than 6 months before the expiration of the certificate and no later than 5 months after the expiration of the certificate.
#*Understand that within the 5 year timeframe, should ICAR suspect a product change that has not been subjected to the tests outlined in this Procedure 5 of Section 10 of the ICAR Guidelines, or any other breach of the conditions described in this chapter, ICAR may take sample products from the market and test their performance against the performance of the certified device.
# Should the manufacturer fail to meet any or all above certificate conditions ICAR may withdraw the certification.
# In disputes regarding the conditions above or the use of a certificate, the decision of ICAR will be binding.
# ICAR will distribute an advice notice regarding any manufacturer distributing RFID devices in conflict with the testing and certification procedures outlined in this Procedure 5 of Section 10 of the ICAR Guidelines.</div>

= Procedure 6: Voluntary sampling of Identification Devices =
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<div>

== Introduction ==
Verification of identification devices is a quality service to ensure ICAR certified devices currently commercially available continue to meet appropriate ISO and ICAR test conformance criteria. ICAR offers this service to national Competent Authorities and other mandated bodies (hereafter referred to as the applicant); however, this service is not offered to manufacturers or their agents. Certification or re-certification of submitted devices cannot occur through the verification service.
Applicants would use this service when there was a concern that an ICAR certified device was failing to meet appropriate ISO and ICAR test conformance criteria. When such a concern exists, the steps in the Device Verification process (see next paragraph) should be enacted.
<div>
== The device verification process ==

# The applicant completes the application form (Appendix D1) and emails it to [mailto:icar@icar.org icar@icar.org].
# The ICAR Secretariat processes the application, provides instructions to the applicant regarding the collection of the samples (as outlined in this Procedure), and issues the invoice.
# The sample devices are collected by the applicant from stock available to the local market. Note: the manufacturer of the device cannot collect or submit the required samples.
# After receipt of the samples by the nominated accredited ICAR laboratory, ICAR will inform the respective manufacturer about the sampling request.
# Devices are tested against the current ICAR standards to determine if the device continues to meet appropriate ISO and ICAR test conformance criteria. The applicant may also request or specify additional testing, provided these tests are defined in other existing ISO or ICAR documented test protocols.
# The test results are compiled into a confidential report. This report is the property of the applicant.
# As part of this testing regime, ICAR will conduct a comparative analysis of the submitted device against the original certification for the same device.
# Once all testing has been completed, the submitted devices will remain at the nominated ICAR accredited laboratory and will not be returned to the applicant.

== Sampling collection protocol ==
Devices shall be collected by the applicant making the request. The sample devices are collected by the applicant from stock available to the local market. The sample devices must be new devices still in their original packaging. It is expected that the devices submitted for testing have been stored in appropriate conditions, i.e., between 5 - 25 degrees Celsius, and the applicant will need to advise the length of time the submitted devices have been in storage. These tags should be shipped to ICAR via a regular logistic mailing service. As stated above, the manufacturer of the device cannot collect or submit the required samples.

The number of device samples required to be submitted for testing is as follows (or as diﬀerently speciﬁed by ICAR):

* For testing of the transponder in RFID devices: 15 RFID devices. If the device is a two-piece device (e.g.), both male and female components must be submitted.
* For testing conventional ear tags or the external material of RFID devices: 135 ear tags. If the device is a two-piece device (e.g.), both male and female components must be submitted.
* Two tag applicators or equivalent devices for the attachment of tags to animals. The applicator must be the applicator specified and sold by the manufacturer for the submitted devices.

After receipt of the samples by the test laboratory but prior to testing, ICAR will inform the respective manufacturer of the sampling request and seek confirmation from the manufacturer that the device obtained for sampling from the specific country or market, is a device that the manufacturer recognises as being or having been marketed or supplied by them in that country. ICAR will also confirm with the manufacturer that the tag applicator supplied by the applicant is the correct applicator for the submitted devices. If the manufacturer states that the devices were not manufactured by them and / or that the supplied applicator is not the correct applicator for the submitted devices, then ICAR shall inform the applicant so that the applicant can choose whether to continue or halt the testing of the devices.

== Testing protocol ==
Device(s) will be tested against the appropriate ICAR Guidelines by an ICAR accredited laboratory nominated by ICAR.

The device test will be carried out by using:

* The Limited Test protocol for the RFID conformance tested devices. (Procedure 1, Section 10 ‘Conformance of Transponders with ISO standards’ available here).
* The Preliminary Assessment protocol for the material performance of the tested devices (Appendix B4. Preliminary Test for Conventional Plastic Ear Tags (here), or Appendix C3. Preliminary Test for External RFID Devices available here).

Additional Testing: The applicant may also request additional testing beyond those specified in the Limited Test protocol or Preliminary Assessment protocol. Provided those additional test(s) are defined in existing ISO or ICAR documented test protocols, the designated test laboratory will provide a quote outlining the additional costs for the requested testing in the test plan they submit to the ICAR Secretariat. The ICAR Secretariat will then advise the applicant of the additional costs for the applicant’s approval..

== Evaluation of tests results ==
As stated above, devices are tested against the current ICAR standards to determine if the device continues to meet appropriate ISO and ICAR test conformance criteria. If the devices meet these criteria, the applicant will be advised accordingly.

During the testing process, ICAR will also conduct a comparative analysis of the submitted device and the original certification for the same device(s). In case of discrepancies between the results and the previous tests, the reference test report will be:

# The full conformance test report, in case of RFID devices.
# The laboratory test report (not the preliminary assessment report), in case of conventional ear tags (or electronic ear tags tested for material and environmental performance).

In reference to composition testing, the IR analysis and the tensile test must be carried out to demonstrate if the material of the current samples is the same as that of the originally tested samples.

If the analysis demonstrates the product has been changed from the originally certified device, ICAR will evaluate those changes. The resulting evaluation may result in ICAR requesting the manufacturer to obtain an appropriate laboratory test of the device to provide a new certification. Appropriate testing for RFIDs can be found in Procedure 1 Section 4.1. Appropriate testing for conventional ear tags can be in Procedure 4 Section 6. If the manufacturer rejects the request of a new test, then the ICAR certification of the device will be revoked..

== Ownership, publication and reporting of results ==
The test results will be compiled into a confidential report, a copy of which will then be transmitted to the applicant who signed the application. The applicant then becomes the owner of that report. The ICAR Secretariat will retain a confidential copy of the report for reference and comparison purposes.

Ownership of the reports:

* Applicant: Owns the sample validation test report but does not have access to any previous reports or reports owned by other applicants.
* Manufacturer: Owns the original test report(s) and can only obtain the sample validation test report upon agreement with the applicant.
* Laboratory: Issues the sample validation test report.
* ICAR:
** Receives the sample validation test report from the laboratory and confirms the devices continue to meet appropriate ISO and ICAR test conformance criteria.
** Conducts a comparative analysis with the original test report to determine if the product has been changed from the originally certified device.

== Confidentiality ==
ICAR will not publish the names of the applicants who have applied for sample validation services nor share the results of tests with the relevant device manufacturers, unless permission is requested and subsequently granted by the applicant.

ICAR will not disclose information about which devices are being or have been tested from which countries/markets without the approval of both the applicant and the manufacturer.

All test results shall be kept confidential between ICAR and the applicant making the request(s). Specific test results are returned only to the applicant making the request(s) and providing the device samples. ICAR strongly requests the test results remain strictly confidential between the applicant, ICAR, and the manufacturer. ICAR strongly discourages the sharing of such reports with any person or any entity other than the aforementioned parties nor should these reports ever be made available to the public domain.[[File:Section 10, Procedure 6, Figure 1.jpg|center|thumb|800x800px|Section 10, Procedure 6, Figure 1]]</div>
</div>
</div>

= Appendix B3: Numbers of Reference Printing =
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<div>
== Values ==
<center>
{| class="wikitable"
|'''Set nr.'''
|'''Digits'''
|-
|1
|8080
|-
|2
|7117
|-
|3
|3883
|-
|4
|5656
|-
|5
|8808
|-
|6
|3383
|-
|7
|1717
|-
|8
|3038
|-
|9
|9989
|-
|10
|4949
|-
|11
|9444
|-
|12
|2727
|-
|13
|2772
|-
|14
|7222
|-
|15
|1441
|-
|16
|1114
|-
|17
|1414
|-
|18
|5665
|-
|19
|6555
|-
|20
|1234
|-
|21
|5678
|-
|22
|9012
|-
|23
|0888
|-
|24
|8998
|-
|25
|8999
|}
</center>
</div>
</div>

= Appendix B4: Preliminary Test for Conventional Plastic Ear Tags =
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== Manufacturer requirements ==
At the commencement of the Preliminary assessment the manufacturer must deliver:

# A sample of 130 ear tags marked with the reference printing applied using the same technique and style as used (or intended to be used) in the commercially marketed tags. Note: Tags used in this phase are likely to be destroyed during testing.
# Two tag applicators or equivalent devices supplied for the application of tags to animals..

== Ear tag design ==
Ear tags shall have smooth, rounded corners and no sharp edges or protrusions especially on the shaft of the piercing pin. The following measurements will be taken:

# The dimensions of the front and rear plate (height, width and thickness).
# The pin (length and diameter).
# The entrance hole of the cap.
# The weight of the complete locked ear tag.
# The distance between the base of the pin and the base of the plate, i.e. the maximum possible distance between the inner side of the male and female components when the device is coupled, measured at the pin, with the two tag plates parallel with each other..

Values and observations potentially impacting on animal welfare will be reported[https://wiki.icar.org/index.php/Section_10_%E2%80%93_Identification_Device_Certification#Annex_A._Applicators_and_insertion_of_ear_tags gs].

== Colour staining test ==
This test detects the risk of potential colour staining resulting from inappropriate production processes with potential toxicity to humans and animals. The test will be done referencing ISO 105-X12. Grey scale will not be assessed but will be replaced by a visual inspection of colour staining on the cloths from the tag component under assessment. The colour staining test will be conducted on three randomly selected ear tags. If any colour residues specific to the colour of the tag are seen on the cloth, then the ear tag has failed the preliminary assessment. The cloth used in this test will be compliant with ISO-105-F09 (Specification for Cotton Rubbing Cloth).

== Locking mechanism checks ==
The primary purpose of these tests is to verify that the male to female locking mechanism, once correctly applied using the supplied applicator, cannot be subsequently dismantled in such a way that would allow the tag or one of the tag parts to be re-used. A locked ear tag must be designed where neither the male nor female part can be re-usable. For one-piece (loop) tags, either the male pin or female locking cap must break such that the tag cannot be used. Tampering with a locked ear tag shall render the tag unusable.

== Pre-conditioning of tag applicator ==
Devices requiring coupling for various testing procedures will be coupled in an environment of 21°C ± 2° and a relative humidity (RH) of 50% ± 10%. Before coupling, the supplied applicator and the devices will be preconditioned to this environment for a minimum period of 24 hours.

== Application test ==
The application evaluation will be carried out using two groups of tags:

Group 1: 80 tags with the front and rear tag components locked together but without being inserted through ears.

Group 2: 40 tags applied and locked into ears obtained post slaughter.

The performance level required for the 120 ear tags shall be:
# Successful locking of the front and rear tag components of all ear tags.
# No breakage of any tag component at locking.
# No deformation of any tag component after locking.
# No unlocking without breakage or irreparable damage to the ear tag..

The test centre will also check the rotation of the tag components on the locked tags. The following characterisation will be used:

# Tag components rotate freely.
# Tag components rotate but not freely.
# Tag components do not rotate.

== Resistance of the locking system ==
The 80 ear tags of Group 1 will be divided into four sub-groups of 20 tags. These four sub-groups will be subjected to increasing forces to determine the force required to cause breakage or unfastening of the ear tag. The forces will be applied at a speed rate of 500 mm/min. The force applied to cause breakage or unfastening of each ear tag will be recorded.

# Group 1: axial test at ambient conditions (21°C ± 2°)
# Group 2: axial test at 55°C (± 2°); the forces will be applied within 10 seconds after the tags are removed from the heating or climatic chamber
# Group 3: transverse test at ambient conditions (21°C ± 2°)
# Group 4: transverse test at 55°C (± 2°); the forces will be applied within 10 seconds after the tags are removed from the heating or climatic chamber.
Transverse testing is not conducted on one-piece (loop) tags.

=== Requirements ===

# None of the ear tags – neither male nor female part – must be re-usable. Male pin tips must break off and remain within the female caps (locking gap).
# At ambient conditions, axially tested tags designed to be used in cattle shall not break with the application of a force lower than 280 Newton.
# At ambient conditions, axially tested tags designed to be used in sheep and / or goats shall not break with the application of a force lower than 200 Newton.
# At ambient conditions, axially tested tags designed to be used in pigs shall not break with the application of a force lower than 200 Newton.

=== Introduction of samples into the climatic chamber ===
To avoid irradiation of the stem and pin, the samples must be closed with the applicator, then pulled apart and turned around so that male faces upwards and the female downwards, or vice versa. See pictures below:
[[File:Imagesample1.png|center|thumb]]
[[File:Imagesample2.png|center|thumb]]
</div>
</div>

= Appendix B5: Laboratory Test for Conventional Plastic Ear Tags =
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== Assessment of descriptive parameters ==
The parameters describing the ear tag will be assessed and compared to the information provided in the Application Form to ensure accuracy of description.

=== Weight and dimensions ===
Ear tags shall have smooth, rounded corners and no sharp edges or protrusions especially on the shaft of the piercing pin. The following measurements will be taken:

# The dimensions of the front and rear plate (height, width and thickness)
# The pin (length and diameter)
# The weight of the complete locked ear tag
# The entrance hole of the cap
# The distance between the base of the pin and the base of the plate, i.e., the maximum possible distance between the inner side of the male and female components when the device is coupled, measured at the pin, with the two tag plates parallel with each other.
The results of these measurements will be compared to the Preliminary Assessment test report to ensure the accuracy of the samples..

=== Composition ===
Because ear tags are attached to "food producing" animals, they must meet specific requirements set down by international laws and regulations. In addition to these requirements, substances affecting animal, human or environmental health need to be detected. As such, certain chemical and physical composition traits of the ear tag will be evaluated. This evaluation will involve 20 ear tags.

==== Characteristics of the ear tag plate plastic ====
To characterise the basic component of the plastic raw material, one ear tag plate is submitted to an Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy analysis. Sample preparation is not necessary as the ear tag plate is pressed directly against the ATR-crystal. After analysis, the resulting ATR spectrum will be compared with characteristic spectra stored in specific databases.

Following this analysis, a material sample is submitted to a Differential Scanning calorimetry (DSC) analysis to analyse the thermal characteristics of the material as per ISO 11357. This analysis allows the detection of overlapping IR curves, e.g. if an additional component of minor quality was used to stretch the main component.

Melting point and glass transition temperatures are recorded to indicate the specific thermal characteristics of the plastic material..

==== Harmful substances ====
Pigmented plastics may contain critical heavy metals which must be recorded. These metals are: Cadmium (Cd), lead (Pb), mercury (Hg) and chromium (Cr). If chromium is detected, an additional analysis of carcinogenic hexavalent chromium will be done. The following limit values must not be exceeded:

# Cadmium: 100 mg/kg
# Lead: 10 mg/kg
# Mercury: 1 mg/kg
# Chromate (Cr VI): < 1 mg/kg

== Pre-conditioning of tag applicator ==
Devices requiring coupling for various testing procedures will be coupled in an environment of 21°C ± 2° and a relative humidity (RH) of 50% ± 10%. Before coupling, the supplied applicator and the devices will be preconditioned to this environment for a minimum period of 24 hours.

== Pre-treatments ==
Various treatments are required to prepare tags for the testing of particular characteristics and are outlined in the following sections. These pre-treatments and ensuing performance assessments are summarized in the following table:

<table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse: collapse; border: medium none; margin-left: 5.4pt" id="table1">
<tr>
<td width="144" valign="top" style="width:108.0pt;border:none;border-top:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">
 </td>
<td colspan="3" valign="bottom" style="width: 181px; border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
New tags</td>
<td colspan="2" valign="bottom" style="width: 161px; border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
UV/rain aged tags</td>
<td width="94" valign="bottom" style="width: 70.2pt; border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
Damp heat/cold aged tags</td>
</tr>
<tr>
<td width="144" valign="top" style="width: 108.0pt; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
 </td>
<td width="91" valign="bottom" style="width: 68.0pt; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
Untreated</td>
<td valign="bottom" style="width: 73px; border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
Acid / Alkaline bath</td>
<td valign="bottom" style="width: 3px; border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm"></td>
<td valign="bottom" style="width: 73px; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
Untreated</td>
<td width="83" valign="bottom" style="width: 62.05pt; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
Abraded</td>
<td width="94" valign="bottom" style="width: 70.2pt; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
Untreated</td>
</tr>
<tr>
<td width="144" valign="top" style="width: 108.0pt; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
Colour Staining</td>
<td width="91" valign="bottom" style="width: 68.0pt; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
X</td>
<td valign="bottom" style="width: 73px; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm"></td>
<td valign="bottom" style="width: 3px; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm"></td>
<td valign="bottom" style="width: 73px; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm"></td>
<td width="83" valign="bottom" style="width: 62.05pt; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm"></td>
<td width="94" valign="bottom" style="width: 70.2pt; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
 </td>
</tr>
<tr>
<td width="144" valign="top" style="width:108.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
Visual readability
Typography
Colour contrast</td>
<td width="91" valign="top" style="width:68.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">

X
X</td>
<td valign="top" style="width: 73px; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm"></td>
<td valign="top" style="width: 3px; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm"></td>
<td valign="top" style="width: 73px; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">

X
X</td>
<td width="83" valign="top" style="width:62.05pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">

X</td>
<td width="94" valign="top" style="width:70.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 </td>
</tr>
<tr>
<td width="144" valign="top" style="width:108.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
Machine readability
Barcode scanning
Barcode quality check</td>
<td width="91" valign="top" style="width:68.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">

X
X</td>
<td valign="top" style="width: 73px; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">

X</td>
<td valign="top" style="width: 3px; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
</td>
<td valign="top" style="width: 73px; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">

X
</td>
<td width="83" valign="top" style="width:62.05pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 
X</td>
<td width="94" valign="top" style="width:70.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 
X</td>
</tr>
<tr>
<td width="144" valign="top" style="width:108.0pt;border:none;border-bottom:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">

Resistance of the locking mechanism</td>
<td width="91" valign="top" style="width:68.0pt;border:none;border-bottom:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">
X</td>
<td valign="top" style="width: 73px; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
</td>
<td valign="top" style="width: 3px; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
</td>
<td valign="top" style="width: 73px; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
X</td>
<td width="83" valign="top" style="width:62.05pt;border:none;border-bottom:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">
 </td>
<td width="94" valign="top" style="width:70.2pt;border:none;border-bottom:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">
X</td>
</tr>
</table></div>
=== Acid and alkaline bath treatment ===
Three coupled ear tags are immersed for 3 weeks in a 21°C acid liquid (acetic acid, pH = 3) and another three coupled ear tags are immersed for 3 weeks in a 21°C alkaline liquid (sodium hydroxide, pH = 12). Both these treatments are to ensure compliance with ISO 175 for thermoplastics and ISO 1817 for vulcanized elastomers. As a reference, another three coupled ear tags are immersed in demineralized water for the same time period as the acid and alkaline baths.

These tests will only be done on ear tags where the tip and inner ring of the locking mechanism contain metal parts to ensure there is no susceptibility to galvanic corrosion.

After treatment, the samples are rinsed with demineralized water and dried for assessment of obvious deformation or material loss. Samples are also weighed before and after tests to detect material loss.

=== Ageing by damp heat and cold ===
In accordance with ISO 4611, 40 ear tags are placed into alternating cycles of 12 hours damp heat (40°C ± 2° / 95% RH) and 12 hours cold (-25°C ± 2°) for a duration of 3 weeks in a climatic chamber.

=== Resistance to artificial ageing ===
In accordance with EN ISO 4892-2, procedure A/cycle 1, 40 coupled ear tags are tested against resistance to sunlight. The exposure chamber will be fitted with xenon-arc lamps according to EN ISO 4892-2 and operated continuously for 1,000 hours. Due to the normal movement of animals' ears, both sides of a coupled ear tag are affected by the sun and other climatic elements. To simulate this process in the laboratory, ear tags undergoing an artificial aging testing within the climatic chamber are turned over after 500 hours. This ensures both sides of the coupled ear tag undergo 500 hours of exposure which has proven to be a sufficient time to assess aging behavior and to measure impacts on the material adequately. These 1,000 hours will consist of repeated cycles of 102 minutes of radiant exposure followed by 18 minutes of combined irradiation and rain simulation. The irradiance level of the xenon-arc lamps will be 60 W/m² (at 300-400 nm).

=== Abrasive treatment ===
Five new, untreated ear tags and five artificially aged ear tags will be subjected to an abrasive treatment as per ISO 9352. These tags will receive 1500 cycles of abrasion in a 21°C ±2° laboratory environment.

The abrasive treatment uses CS17 abrasive wheels and a load of 1,000 g (or 9.8 N). The front plates of the tags are cut to a disc of about 100 mm in diameter and mounted on the test plate of the Taber Abrader.

=== Colour staining test ===
The test consists in wiping the ear tags with soft white tissue. If colour is visible on the tissue, the test is a fail.

=== Introduction of samples into the climatic chamber ===
To avoid irradiation of the stem and pin, the samples must be closed (coupled) with the manufacturer-supplied applicator, then spread apart and turned around so that male faces upwards and the female downwards, or vice versa. See pictures below:
[[File:Imagesample1.png|center|thumb]]
[[File:Imagesample2.png|center|thumb]]

== Performance Assessment ==

=== Typography readability ===
Five new, untreated tags and five tags from the following two treatment groups will be selected for assessment:

# Group 1: Artificially aged tags not subjected to the abrasive treatment.
# Group 2: Artificially aged tags subjected to the abrasive treatment.

Five randomly chosen numbers as given in [[Section 10 – Identification Device Certification#Appendix B3: Numbers of Reference Printing|Appendix B3]] will be printed on five white pages of paper. The font size, print style and character spacing will replicate that used for the ear tags.

The test tags and the pages with the printed numbers will be placed on a vertical surface (viewing surface) at head height in an appropriately lit laboratory room. Five assessors will stand 15 metres from the viewing surface and then commence walking towards it. Each assessor will attempt to read the numbers on the different ear tags and pages and the distance at which each device (ear tag or page) can be read without error will be recorded on the evaluation sheet.

The mean reading distance for both the pages and the ear tags will be separately calculated for each assessor and for the average of the assessors.

The following requirements must be met:
# New, untreated tags: The mean distance at which the reference printing is read on the ear tags must be at least 80 % of the mean distance at which the pages are read.
# Artificially aged tags with and without the abrasive treatment: The mean distance at which the reference printing is read for the ear tags must be at least 65 % of the mean distance at which the pages are read..

=== Evaluation of colour contrast change ===
The colour difference of the ear tag plates and of the laser printing is measured and compared between three new ear tags and three artificially aged ear tags by use of spectral photometric measuring equipment according to ISO 11664-4.

After artificial ageing, the change in colour must be less than delta E* of 15 CIELAB units.

=== Evaluation of contrast change for ear tags with combined laser and inkjet printing ===
Combined laser and inkjet printed tags can be tested as an additional option. If the option is chosen, then they will be tested in addition and parallel to laser printed tags only. If combined laser and inkjet printed tags will be tested, then ten tags with combined laser and inkjet printing shall be delivered in addition to the standard required quantity of laser printed tags for laboratory testing.

The tags printed with combined laser and inkjet printing will be subjected to the colour contrast change evaluation test before and after an abrasive treatment (see Appendix B5 Paragraph 3.5). The comparative evaluation will be against a new tag printed with laser only. The change in colour must not fade beyond a maximum colour change of delta E* ≤ 15 CIELAB units.

=== Evaluation of machine readability (optional) ===
This evaluation will occur if the manufacturer requests the machine readability testing in the Application Form ([http://old.icar.org/wp-content/uploads/2017/04/Annex-B1-updated-March-2017.pdf Appendix B1]).

For ear tags with linear barcodes, the "Quiet Zone" or margin at each end of the barcode must be at least 5mm. The height of the barcode must be at least 8mm.

==== Barcode scanning ====
The ear tags subjected to the Phase 3 treatments will be scanned with three different handheld barcode readers.

The treated ear tags will be scanned in sequence and after the initial ear tag is successfully read, the second tag is scanned until successfully read. Each ear tag will be scanned a maximum of four times. This procedure is repeated for each tag in the treatment group and after the last tag is scanned, the scanning is recommenced (Run 2) with the first tag. A total of 60 scans per treatment and reader type will be conducted to obtain sufficient data to assess performance.

The number of scans required to successfully read each tag (e.g. one, two, three or four) in each run is recorded.

The scanning success rate of tags from each treatment group is expressed in a percentage value and based on the number of scans required for a successful read. The performance of the tag is assessed against the minimum performance standards shown below::<div align="center">
<table class="MsoNormalTable" border="0" cellspacing="0" cellpadding="0" style="border-collapse: collapse" id="table1">
<tr style="page-break-inside: avoid">
<td valign="bottom" style="border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
No. of scans required</td>
<td valign="bottom" style="border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
Proportion of tags successfully read at each scan</td>
</tr>
<tr style="page-break-inside: avoid">
<td valign="top" style="border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
1</td>
<td valign="top" style="border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
95%</td>
</tr>
<tr style="page-break-inside: avoid">
<td valign="top" style="padding:0cm 5.4pt 0cm 5.4pt">
2</td>
<td valign="top" style="padding:0cm 5.4pt 0cm 5.4pt">
98%</td>
</tr>
<tr style="page-break-inside: avoid">
<td valign="top" style="border:none;border-bottom:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">
3</td>
<td valign="top" style="border:none;border-bottom:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">
99.7%</td>
</tr>
</table>
</div>

The scanning performance achieved for each treatment is included in the ICAR report sent to Service-ICAR at the conclusion of the laboratory tests.

==== Barcode print quality assessment ====
Print quality assessment will be undertaken on ten new, untreated tags using the protocols described below.

Using an ISO 15426-1 barcode verifier the linear barcodes are assessed for print quality according to ISO 15416. Every ear tag will be scanned ten times to build average grades.

An ANSI scale of A (highly satisfactory) to F (unsatisfactory) will be used to grade the print quality for each characteristic. When determining the overall print quality, the final score for the code on a tag is the worst grade recorded for any of the assessed characteristics. Failure reasons will be given in the test report.

The following linear barcode print quality specifications must be met:
# Decode (the only grades used are A and F): A
# Decodability: minimum D meaning >25%
# Check Character (if available): OK
# Symbol Contrast (Rmax - Rmin): minimum D meaning >25%

In the print contrast component of the test for 2D barcodes, the QR code and DM symbols are assessed for print quality using a barcode verifier that complies with the AIM International standards, Section "M" under Matrix Code Print Quality Guideline.

The following 2D barcode print quality specifications must be met:
# Decode (the only grades used are A and F): A
# Symbol contrast: minimum D, meaning >25%
# Print growth X axis and Y axis: A/A, A/B or B/A
# Axial non-uniformity: A

The standard of symbols must be no more than one print quality grade below that for each parameter on an unused tag without treatment.

==== Evaluation of the resistance of the locking system ====
The ear tags are pre-conditioned for at least 2 hours before testing at the respective temperature. Testing must occur within 10 seconds after the ear tag is removed from the chamber.

30 new, untreated ear tags, 30 artificially aged ear tags and 30 ear tags submitted to the damp heat and cold treatment will be subjected to increasing forces to determine the force required to cause breakage or unfastening of the ear tag.

For cattle, sheep and goat ear tags, the test is performed at -25°C (± 2°), 21°C (±2°C) and 55°C (± 2°C) combined with 50% RH (when the temperature is higher than 0°C) with 10 ear tags from the three treatment variations.

For pig ear tags, the test is performed at -10°C (± 2°), 21°C (± 2°) and 55 °C (± 2°) combined with 50% RH (when the temperature is higher than 0°C) with 10 ear tags from the three treatment variations.

For tags which are used for both sheep/goat and pigs, the test is performed at -25°C (±2°), ‑10°C (± 2°), 21°C (± 2°C), and 55°C (± 2°C) combined with 50% RH (when the temperature is higher than 0°C) with 10 ear tags from the three treatment variations.

The forces will be applied at a rate of 500 mm/min within 10 seconds after the tags are removed from the climatic chamber. The force applied to cause breakage or unfastening of each ear tag will be recorded.

==== Requirements ====
# None of the ear tags – neither male nor female part – must be re-usable. Male pin tips must break off and remain within the female caps (locking gap)
# At 21°C (± 2°C), no breakage should occur in:
#* Tags designed to be used in cattle with the application of a force lower than 280 Newton.
#* Tags designed to be used in sheep and / or goats with the application of a force lower than 200 Newton.
#* Tags designed to be used in pigs with the application of a force lower than 200 Newton.
# The minimum breaking force applies to devices irrespective of treatments (artificial ageing, damp heat and cold).

Additionally, the distortion occurring in the ear tag at the time of breakage or unfastening will be recorded during the tensile tests as an indicator for any changes in the mechanical properties of the plastic after exposure to the artificial ageing and the damp heat/cold treatments.
----[1] For ear tags made of moisture-sensitive material like PA (polyamide), the test must be carried out at the same laboratory humidity (50 % ± 10 %) like used during the preconditioning.
</div>

= Appendix C3: Preliminary Test for External RFID Devices =
<div class="mw-collapsible mw-collapsed">
<div>
== Manufacturer requirements ==
At the commencement of the Preliminary Assessment the manufacturer must deliver
# A sample of 130 RFID devices programmed with the reference ID codes and the reference printing. The printing must be applied using the same technique and style as used (or intended to be used) in the commercially marketed devices. Note: Devices used in this phase are likely to be destroyed during testing.
# Two applicators or equivalent devices supplied for the application of devices to animals.

== Ear tag design ==
Ear tags shall have smooth, rounded corners and no sharp edges or protrusions especially on the shaft of the piercing pin. The following measurements will be taken:

# The dimensions of the front and rear plate (height, width and thickness).
# The pin (length and diameter).
# The entrance hole of the cap.
# The weight of the complete locked ear tag.
# The distance between the base of the pin and the base of the plate, i.e., the maximum possible distance between the inner side of the male and female components when the device is coupled, measured at the pin, with the two tag plates parallel with each other.

Values and observations potentially impacting on animal welfare will be reported.

== Colour staining test ==
This test detects the risk of potential colour staining resulting from inappropriate production processes with potential toxicity to humans and animals. The test will be done referencing ISO 105-X12. Grey scale will not be assessed but will be replaced by a visual inspection of colour staining on the cloths from the tag component under assessment. The colour staining test will be conducted on three randomly selected ear tags. If any colour residues specific to the colour of the tag material are seen on the cloth, then the ear tag has failed the preliminary assessment. The cloth used in this test will be compliant with ISO-105-F09 (Specification for Cotton Rubbing Cloth).

== Electronic readability check ==
Every submitted RFID ear tag will be read with an ICAR approved handheld reader to ensure the reference ID codes transmitted meet the requirements outlined in section 4.5 of Procedure 5, Section 10 ‘Testing of External RFID Devices’ (available [http://www.icar.org/Guidelines/10-Procedure-5-Testing-of-External-RFID-Devices.pdf here]).

== Locking mechanism checks ==
The primary purpose of these tests is to verify that the male to female locking mechanism, once correctly applied using the supplied applicator, cannot be subsequently dismantled in such a way that would allow the tag, or one of the tag parts, to be re-used. A locked ear tag must be designed where neither the male nor female part can be re-usable. For one-piece (loop) tags, either the male pin or female locking cap must break such that the tag cannot be used. Tampering with a locked ear tag shall render the tag unusable.

== Pre-conditioning of tag applicator ==
Devices requiring coupling for various testing procedures will be coupled in an environment of 21°C ± 2° and a relative humidity (RH) of 50% ± 10%. Before coupling, the supplied applicator and the devices will be preconditioned to this environment for a minimum period of 24 hours.

== Application test ==
The application evaluation will be carried out using two groups of tags:

# '''RFID ear tags classified as flag tags (extended front plates):'''
#* Group 1: 80 tags with the front and rear tag components locked together but without being inserted through ears
#* Group 2: 40 tags applied and locked into ears obtained post slaughter
# '''RFID ear tags not classified as flag tags:'''
#* Group 1: 40 tags with the front and rear tag components locked together but without being inserted through ears
#* Group 2: 40 tags applied and locked into ears obtained post slaughter

The performance level required for the submitted ear tags shall be:

# Successful locking of the front and rear tag components of all ear tags.
# No breakage of any tag component at locking.
# No deformation of any tag component after locking.
# No unlocking without breakage or irreparable damage to the ear tag.

The test centre will also check the rotation of the tag components on the locked tags. The following characterisation will be used:

# Tag components rotate freely.
# Tag components rotate but not freely.
# Tag components do not rotate..

== Resistance of the locking system ==
The ear tags must be pre-conditioned for at least 2 hours before testing at the respective temperature. Testing must occur within 10 seconds after the ear tag is removed from the climatic chamber.

=== Flag Tags ===
The 80 RFID ear tags of Group 1 will be divided into four sub-groups of 20 tags. Those four sub‑groups will be subjected to increasing forces to determine the force required to cause breakage or unfastening of the ear tag. The forces will be applied at a speed rate of 500 mm/min. The force applied to cause breakage or unfastening of each ear tag will be recorded.

# Group 1: axial test at ambient conditions 21°C (± 2°).
# Group 2: axial test at 55°C (± 2°); the forces will be applied immediately after the tags are removed from the heating or climatic chamber.
# Group 3: transverse test at ambient conditions 21°C (± 2°).
# Group 4: transverse test at 55 °C (± 2°); the forces will be applied within 10 seconds after the tags are removed from the heating or climatic chamber..

'''Requirements'''

# None of the ear tags - neither male nor female part - must be re-usable. Male pin tips must break off and remain within the female caps (locking gap).
# At ambient conditions, axially tested tags designed to be used in cattle shall not break with the application of a force lower than 280 Newton.
# At ambient conditions, axially tested tags designed to be used in sheep and / or goats shall not break with the application of a force lower than 200 Newton.
# At ambient conditions, axially tested tags designed to be used in pigs shall not break with the application of a force lower than 200 Newton.

=== Ear tags not classified as flag tags ===
The ear tags must be pre-conditioned for at least 2 hours before testing at the respective temperature. Testing must occur within 10 seconds after the ear tag is removed from the climatic chamber.

The 40 RFID ear tags of Group 1 will be divided into two sub-groups of 20 tags. Those two sub‑groups will be subjected to increasing forces to determine the force required to cause breakage or unfastening of the ear tag. The forces will be applied at a speed rate of 500 mm/min. The force applied to cause breakage or unfastening of each ear tag will be recorded.
# Group 1: axial test at ambient conditions 21°C (± 2°).
# Group 2: axial test at 55°C (± 2°); the forces will be applied immediately after the tags are removed from the heating or climatic chamber.

'''Requirements'''

# None of the ear tags - neither male nor female part - must be re-usable. Male pin tips must break off and remain within the female caps (locking gap).
# At ambient conditions, axially tested tags designed to be used in cattle shall not break with the application of a force lower than 280 Newton.
# At ambient conditions, axially tested tags designed to be used in sheep and / or goats shall not break with the application of a force lower than 200 Newton.
# At ambient conditions, axially tested tags designed to be used in pigs shall not break with the application of a force lower than 200 Newton.

==== Introduction of samples into the climatic chamber ====
To avoid irradiation of the stem and pin, the samples must be closed with the applicator, then pulled apart and turned around so that male faces upwards and the female downwards, or vice versa. See pictures below:
[[File:Imagesample1.png|center|thumb]]
[[File:Imagesample2.png|center|thumb]]

== Testing RFID leg tags ==
To assess conformance of the RFID leg tags with the information given in the application form and to also detect any major failure e.g. electronic non-readability, damage of the device at application, inappropriate animal welfare design etc., the leg tags will be submitted to a Preliminary Assessment.

=== Leg tag design ===
RFID leg tags shall have smooth, rounded corners and no sharp edges or protrusions. The following measurements will be taken:

# The weight of the leg tag
# The dimensions of the leg tag (length, width and thickness)
# The adjustable diameter

Values and observations potentially impacting on animal welfare will be reported.

=== Electronic readability check ===
Every submitted RFID leg tag will be read with an ICAR approved handheld reader to ensure the reference ID codes transmitted meet the requirements outlined in section 4.5 of Procedure 5, Section 10 ‘Testing of External RFID Devices’ (available [http://www.icar.org/Guidelines/10-Procedure-5-Testing-of-External-RFID-Devices.pdf here]).

</div>
</div>

= Appendix C4: Laboratory Test for External RFID Devices =
<div class="mw-collapsible mw-collapsed">
<div>
== Assessment of descriptive parameters ==
The parameters describing the RFID device will be assessed and compared to the information provided in the Application Form to ensure accuracy of description.

=== Weight and dimensions ===
The following measurements will be taken from five of the submitted RFID devices:

# Ear tags shall have smooth, rounded corners and no sharp edges or protrusions especially on the shaft of the piercing pin. The following measurements will be taken:
#* The dimensions of the front and rear plate (height, width and thickness)
#* The pin (length and diameter)
#* The weight of the complete locked ear tag
#* The entrance hole of the cap
#* The distance between the base of the pin and the base of the plate, i.e., the maximum possible distance between the inner side of the male and female components when the device is coupled, measured at the pin, with the two tag plates parallel with each other..
# Leg tags shall have smooth, rounded corners and no sharp edges or protrusions. The following measurements will be taken:
#* The weight of the leg tag.
#* The dimensions of the leg tag (length, width and thickness).
#* The adjustable diameter.

The results of these measurements will be compared to the Preliminary Assessment test report to ensure the accuracy of the samples.

=== Composition ===
Because RFID devices are attached to "food producing" animals, they must meet specific requirements set down by international laws and regulations. In addition to these requirements, substances affecting animal, human or environmental health need to be detected. As such, certain chemical and physical composition traits of the RFID device will be evaluated. This evaluation will involve 20 RFID devices.

==== Characteristics of the plastic of the ear or leg tag ====
To characterise the basic component of the plastic raw material, one device is submitted to an Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy analysis. If the RFID ear tag contains a flag (an extended plate), the ear tag plate is pressed directly against the ATR-crystal. With leg tags or ear tags without a flag, the laboratory will determine if sample preparation is necessary. After analysis, the resulting ATR spectrum will be compared with characteristic spectra stored in specific databases.

Following this analysis, a material sample is submitted to a Differential Scanning calorimetry (DSC) analysis to analyse the thermal characteristics of the material as per according to ISO 11357. This analysis allows the detection of overlapping IR curves, e.g. if an additional component of minor quality was used to stretch the main component.

Melting point and glass transition temperatures are recorded to indicate the specific thermal characteristics of the plastic material.

==== Harmful substances ====
Pigmented plastics may contain critical heavy metals which must be recorded. These metals are: Cadmium (Cd), lead (Pb), mercury (Hg) and chromium (Cr). If chromium is detected, an additional analysis of carcinogenic hexavalent chromium will be done. The following limit values must not be exceeded:

# Cadmium: 100 mg/kg
# Lead: 10 mg/kg
# Mercury: 1 mg/kg
# Chromate (Cr VI): < 1 mg/kg

== Pre-conditioning of tag applicator ==
Devices requiring coupling for various testing procedures will be coupled in an environment of 21°C ± 2° and a relative humidity (RH) of 50% ± 10%. Before coupling, the supplied applicator and the devices will be preconditioned to this environment for a minimum period of 24 hours.

== Performance Assessments ==
The tests described in this section are designed to determine the stability and endurance of the RFID devices. The performance assessments are summarized in the following table.

'''Note''': Acid and alkaline treatments are mandatory in ear tags where the tip and inner ring of the locking mechanism contain metal parts.<div align="center">
<table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse: collapse; border: medium none; margin-left: 5.4pt" id="table1">
<tr>
<td width="239" valign="bottom" style="width:179.1pt;border:none;border-top:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">
 </td>
<td colspan="3" width="255" valign="bottom" style="width: 191.15pt; border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
Electronic ear tags</td>
<td width="95" valign="bottom" style="width: 70.9pt; border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
Electronic leg tags</td>
</tr>
<tr>
<td width="239" valign="bottom" style="width: 179.1pt; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
 </td>
<td width="48" valign="bottom" style="width: 36.0pt; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
New</td>
<td width="103" valign="bottom" style="width: 77.2pt; border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
Artificially aged</td>
<td width="104" valign="bottom" style="width: 77.95pt; border-left: medium none; border-right: medium none; border-top: 1.0pt solid windowtext; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
Damp heat treated</td>
<td width="95" valign="bottom" style="width: 70.9pt; border-left: medium none; border-right: medium none; border-top: medium none; border-bottom: 1.0pt solid windowtext; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
New</td>
</tr>
<tr>
<td width="239" style="width: 179.1pt; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">

Artificial ageing (ISO 4892-2, A/1)</td>
<td width="48" valign="top" style="width: 36.0pt; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
X</td>
<td width="103" valign="top" style="width: 77.2pt; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
 </td>
<td width="104" valign="top" style="width: 77.95pt; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
 </td>
<td width="95" valign="top" style="width: 70.9pt; border: medium none; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm">
X</td>
</tr>
<tr>
<td width="239" style="width:179.1pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">

Free fall (IEC 60068-2-32)</td>
<td width="48" valign="top" style="width:36.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
X</td>
<td width="103" valign="top" style="width:77.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
X</td>
<td width="104" valign="top" style="width:77.95pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 </td>
<td width="95" valign="top" style="width:70.9pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
X</td>
</tr>
<tr>
<td width="239" style="width:179.1pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">

Cold (IEC 60068-2-1)</td>
<td width="48" valign="top" style="width:36.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
X</td>
<td width="103" valign="top" style="width:77.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 </td>
<td width="104" valign="top" style="width:77.95pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 </td>
<td width="95" valign="top" style="width:70.9pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
X</td>
</tr>
<tr>
<td width="239" style="width:179.1pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">

Dry heat (IEC 60068-2-2)</td>
<td width="48" valign="top" style="width:36.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
X</td>
<td width="103" valign="top" style="width:77.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 </td>
<td width="104" valign="top" style="width:77.95pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 </td>
<td width="95" valign="top" style="width:70.9pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
X</td>
</tr>
<tr>
<td width="239" style="width:179.1pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">

Damp heat (ISO 4611)</td>
<td width="48" valign="top" style="width:36.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
X</td>
<td width="103" valign="top" style="width:77.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 </td>
<td width="104" valign="top" style="width:77.95pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 </td>
<td width="95" valign="top" style="width:70.9pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
X</td>
</tr>
<tr>
<td width="239" style="width:179.1pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">

Tensile test of the locking mechanism</td>
<td width="48" valign="top" style="width:36.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
X</td>
<td width="103" valign="top" style="width:77.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
X</td>
<td width="104" valign="top" style="width:77.95pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
X</td>
<td width="95" valign="top" style="width:70.9pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 </td>
</tr>
<tr>
<td width="239" style="width:179.1pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">

Visual readability
Typography (flag tags only)
Colour contrast change</td>
<td width="48" valign="top" style="width:36.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 
X
X</td>
<td width="103" valign="top" style="width:77.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 
X
X</td>
<td width="104" valign="top" style="width:77.95pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 </td>
<td width="95" valign="top" style="width:70.9pt;border:none;padding:0cm 5.4pt 0cm 5.4pt">
 </td>
</tr>
<tr>
<td width="239" style="width:179.1pt;border:none;border-bottom:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">

Electronic readability (ISO 24631-1, ISO 24631-3)*</td>
<td width="48" valign="top" style="width:36.0pt;border:none;border-bottom:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">
X</td>
<td width="103" valign="top" style="width:77.2pt;border:none;border-bottom:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">
X</td>
<td width="104" valign="top" style="width:77.95pt;border:none;border-bottom:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">
X</td>
<td width="95" valign="top" style="width:70.9pt;border:none;border-bottom:solid windowtext 1.0pt;
padding:0cm 5.4pt 0cm 5.4pt">
X</td>
</tr>
</table>
</div>

A readability test is performed after every environmental test.

=== Initial readability test ===
20 randomly selected RFID devices will be read before commencing any environmental test. The readability test is done according to ISO 24631-1 and 24631-3. Identification number (ID code), resonance frequency, minimum activation field strength and all relevant performance parameters are measured and recorded. The recorded values will be used as the reference for every following read test.

=== Acid and alkaline bath treatment ===
Three coupled ear tags are immersed for 3 weeks in a 21°C acid liquid (acetic acid, pH = 3) and another three coupled ear tags are immersed for 3 weeks in a 21°C alkaline liquid (sodium hydroxide, pH = 12). Both these treatments are to ensure compliance with ISO 175 for thermoplastics and ISO 1817 for vulcanized elastomers. As a reference, another three coupled ear tags are immersed in demineralized water for the same time period as the acid and alkaline baths.

These tests will only be done on ear tags where the tip and inner ring of the locking mechanism contain metal parts to ensure there is no susceptibility to galvanic corrosion.

After treatment, the samples are rinsed with demineralized water and dried for assessment of obvious deformation or material loss. Samples are also weighed before and after tests to detect material loss.

=== Resistance to artificial ageing ===
In accordance with EN ISO 4892-2, procedure A/cycle 1, 40 coupled ear tags are tested against resistance to sunlight. The exposure chamber will be fitted with xenon-arc lamps according to EN ISO 4892-2 and operated continuously for 1,000 hours. Due to the normal movement of animals' ears, both sides of a coupled ear tag are affected by the sun and other climatic elements. To simulate this process in the laboratory, ear tags undergoing an artificial aging testing within the climatic chamber are turned over after 500 hours. This ensures both sides of the coupled ear tag undergo 500 hours of exposure which has proven to be a sufficient time to assess aging behavior and to measure impacts on the material adequately. These 1,000 hours will consist of repeated cycles of 102 minutes of radiant exposure followed by 18 minutes of combined irradiation and rain simulation. The irradiance level of the xenon-arc lamps will be 60 W/m² (at 300-400 nm).

=== Resistance to tensile loading ===
This test only applies to RFID ear tags (not applicable for leg tags).

The ear tags are pre-conditioned for at least 2 hours before testing at the respective temperature. Testing must occur within 10 seconds after the ear tag is removed from the climatic chamber.

This test is done using 30 new ear tags, 30 artificially aged tags and 30 tags submitted to damp heat treatment. For cattle, sheep, and goat ear tags, the test is performed at -25°C (± 2°), 21°C (±2°C) and 55°C (± 2°C) combined with 50% RH (when the temperature is higher than 0°C) with 10 ear tags from the three treatment variations.

'''Note''': For ear tags made of moisture-sensitive material like PA (polyamide), the test must be carried out at the same laboratory humidity (50 % ± 10 %) as used during preconditioning.

For pig ear tags the test is performed at -10°C (± 2°), 21°C (± 2°) and 55 °C (± 2°) combined with 50% RH (when the temperature is higher than 0°C) with 10 ear tags from the three treatment variations.

For tags which are used for both sheep/goat and pigs, the test is performed at -25°C (±2°), ‑10 °C (± 2°), 21°C (± 2°C), and 55°C (± 2°C).

To test the tensile strength of the locking mechanism the ear tag is affixed to a test jig simulating its application and attempts are made to remove the ear tag by subjecting it to increasing forces. The class 1 tensile test machine shall operate at a speed rate of 500 mm/min and be capable of generating loads of up to 1,000 N.
An increasing load will be applied in axial direction. The maximum load and the effect(s) of the tensile force on the appearance and/or efficacy of the ear tags will be recorded.

'''Requirements'''

# None of the ear tags – neither male nor female part – must be re-usable. Male pin tips must break off and remain within the female caps (locking cap).
# At ambient conditions (21°C ± 2°), ear tags designed to be used in cattle shall not break with application of a force lower than 280 Newton.
# At ambient conditions (21°C ± 2°), ear tags designed to be used in sheep and / or goats shall not break with the application of a force lower than 200 Newton.
# At ambient conditions (21°C ± 2°), ear tags designed to be used in pigs shall not break with the application of a force lower than 200 Newton.
# The minimum breaking force applies to devices irrespective of treatments (artificial aging, damp heat, etc.)

=== Resistance to impact of free fall ===
When tested in accordance with IEC 60068-2-32 the RFID device shall not split or crack after falling 1000 mm onto a concrete surface. The test conditions are as follows:

# The tag component containing the transponder is levelled in 3 attitudes (horizontally, vertically top and bottom) and dropped twice in each attitude.
# The above test is carried out on three new and three artificially aged devices.
# The test shall be carried out at a temperature of 21°C (± 3°) and at ambient humidity. The test is repeated after an hour's storage at -20°C (± 2°) immediately after the device is removed from the climatic chamber.

After the free fall test, a readability test is performed according to ISO 24631-1 and ISO 24631‑3 on the tested RFID devices to ensure every device has survived the procedure with the transponder in situ and remains compliant with ISO 11784 and ISO 11785. The measured values are compared to those of the reference devices.

=== Resistance to cold ===
In accordance with IEC 60068-2-1, 10 new tags are exposed to a constant climate of -25°C (± 2°) for 24 hours.

Directly after removing the samples from the climatic chamber a readability test is performed according to ISO 24631-1 and ISO 24631-3 on the tested RFID devices to ensure every device has survived the procedure with the transponder in situ with no change in performance. The measured values are compared to those of the reference devices.

== Resistance to dry heat ==
In accordance with IEC 60068-2-2, 10 new tags are exposed to a constant climate of 55°C (± 3°) for 24 hours.

Directly after removing the samples from the climatic chamber a readability test is performed according to ISO 24631-1 and ISO 24631-3 on the tested RFID devices to ensure every device has survived the procedure with the transponder in situ with no change in performance. The measured values are compared to those of the reference devices.

=== Resistance to damp heat and cold ===
In accordance with ISO 4611, 40 ear tags are placed into alternating cycles of 12 hours damp heat (40°C ± 2° / 95% RH) and 12 hours cold (-25°C ± 2°) for a duration of 3 weeks in a climatic chamber.

Upon completion of this test, a readability test is performed on 10 ear tags according to ISO 24631‑1 and ISO 24631-3 on the tested RFID devices to ensure every device has survived the procedure with the transponder in situ with no change in performance. The measured values are compared to those of the initial test.

=== Introduction of samples into the climatic chamber ===
To avoid irradiation of the stem and pin, the samples must be closed (coupled) with the manufacturer-supplied applicator, then spread apart and turned around so that male faces upwards and the female downwards, or vice versa. See pictures below:
[[File:Imagesample1.png|center|thumb]]
[[File:Imagesample2.png|center|thumb]]
=== Typography readability ===
This test applies to RFID ear tags classified as flag tags only.

Five new ear tags and five artificially aged tags will be selected for assessment.

Five randomly chosen numbers as given in [[Section 10 – Identification Device Certification#Appendix B3: Numbers of Reference Printing|Appendix B3]] will be printed on five white pages of paper. The font size, print style and character spacing will replicate that used for the ear tags.

The test tags and the pages with the printed numbers will be placed on a vertical surface (viewing surface) at head height in an appropriately lit laboratory room. Five assessors will stand 15 metres from the viewing surface and then commence walking towards it. Each assessor will attempt to read the numbers on the different ear tags and pages and the distance at which each device (ear tag or page) can be read without error will be recorded on the evaluation sheet.

The mean reading distance for both the pages and the ear tags will be separately calculated for each assessor and for the average of the assessors.

The following requirements must be met:
# New, untreated tags: The mean distance at which the reference printing is read on the ear tags must be at least 80% of the mean distance at which the pages are read.
# Artificially aged tags: The mean distance at which the reference printing is read for the ear tags must be at least 65% of the mean distance at which the pages are read.

=== Evaluation of colour contrast change ===
The colour difference of the ear tag plates and of the laser printing is measured and compared between three new ear tags and three artificially aged ear tags by use of spectral photometric measuring equipment according to ISO 11664-4.

After artificial ageing, the change in colour must be less than delta E* of 15 CIELAB units.

=== Evaluation of contrast change for ear tags with combined laser and inkjet printing ===
Combined laser and inkjet printed tags can be tested as an additional option. If the option is chosen, then they will be tested in addition and parallel to laser printed tags only. If combined laser and inkjet printed tags will be tested, then ten tags with combined laser and inkjet printing shall be delivered in addition to the standard required quantity of laser printed tags for laboratory testing.

The tags printed with combined laser and inkjet printing will be subjected to the colour contrast change evaluation test before and after an abrasive treatment (see Appendix B5 Paragraph 3.5). The comparative evaluation will be against a new tag printed with laser only. The change in colour must not fade beyond a maximum colour change of delta E* ≤ 15 CIELAB units.
</div>
</div>

User:Bgolden/sandbox

2025-09-03T10:02:38Z

Bgolden:

<big><big>Term:</big></big> 
<blockquote>
<big>Accuracy</big>
</blockquote>

<big><big>Abbreviation:</big></big> 
-none-

<big><big>Definition:</big></big> 
extent of correctness of an estimate obtained with an analytical method, also called overall accuracy. It is expressed through a standard deviation that combines both random error (precision) and systematic error of the method. The part independent from calibration and precision errors, so-called ‘accuracy of estimate’, is a characteristic of alternative methods of analysis.

<big><big>Reference:</big></big> 
ICAR Guideline

under development - section 12

(entry: 561) 

This is a test of visual editing
Now erase it

<center>
[[File: Messages_0_(8).mp4 ]]
</center>
=Blood cards=
<youtube>MSaY2o5CUck</youtube>
=Test=
<youtube>V9pTlgyWsx0</youtube>
=Anoter test=
<youtube>PxKmxKVvVEA?si=C6x0keKAvgU009Da</youtube>

User:Bgolden/sandbox

2025-09-01T16:39:42Z

Bgolden:

User:Bgolden/sandbox

2025-09-01T16:05:16Z

Bgolden:

User:Bgolden/sandbox

2025-09-01T16:02:10Z

Bgolden:

User:Bgolden/sandbox

2025-09-01T15:57:52Z

Bgolden:

File:Messages 0 (8).mp4

2025-09-01T15:56:01Z

Bgolden: test of video

== Summary ==
test of video

Section 20: Beef

2025-08-04T13:02:22Z

Bgolden:

Section 20: Activities

2025-05-13T12:06:04Z

Bgolden:

Section 20: Completed activities

2025-05-09T13:46:51Z

Bgolden:

Section 20: Laser Methane Detector

2025-05-02T10:48:55Z

Bgolden:

Section 20: Greenfeed SOP

2025-05-02T10:48:23Z

Bgolden:

Greenfeed

2025-05-02T10:47:44Z

Bgolden:

Section 20: Respiration chamber

2025-05-02T10:47:23Z

Bgolden:

Section 20: Portable Accumulation Chamber

2025-05-02T10:46:52Z

Bgolden:

Section 20: Wearables

2025-05-02T10:46:21Z

Bgolden:

Breath sampling during milk and feeding

2025-05-02T10:45:21Z

Bgolden:

Section 20: Sniffer SOP

2025-05-02T10:44:51Z

Bgolden:

Section 20: Sniffer SOP

2025-05-02T10:44:25Z

Bgolden:

Breath sampling during milk and feeding

2025-05-02T10:44:01Z

Bgolden:

Section 20: Wearables

2025-05-02T10:43:34Z

Bgolden:

Section 20: Portable Accumulation Chamber

2025-05-02T10:43:03Z

Bgolden:

Section 20: Respiration chamber

2025-05-02T10:41:51Z

Bgolden:

Section 20: Respiration chamber

2025-05-02T10:41:34Z

Bgolden:

Approval of Page Process

2025-04-25T14:21:47Z

Bgolden:

[[Category: SOP]]

'''PURPOSE'''

Description of the procedures for production, revision and editing of the ICAR Guidelines Wiki implementation.

'''SCOPE'''

Coordinating the ICAR Team in producing, maintaining and editing of the proper style, and organisation of the ICAR Guidelines Wiki implementation.

'''PROJECT TEAM'''

'''Chief Executive'''

'''Working Groups''': The groups designated to be responsible for drafting, and editing Guidelines Wiki Sections’ content.

'''Sub-Committee''': Any part of a Working Group designated as a content creator for a portion of the Guidelines Wiki Section.

'''Administrator:''' individual(s) whose Wiki account has all management permissions enabled.

'''PROCEDURES & RESPONSIBILITIES'''

* The production of the Guidelines Wiki content is the responsibility of each Working Group and Sub-Committee.

* The Administrator will follow the development of the Guidelines Wiki and the meetings of the Sub-Committee and Working Group for their production and/or editing.

* For equations, the Sub-Committee and Working Group will provide the reference for any future evaluation of the correctness of the equation reported in the Guidelines.

* The Working Group or Sub-Committee Chair will designate at least two members (Authors) to the Administrator. These Authors will be given accounts with permission to modify and add text to only their specific Guidelines Wiki Section. Other members will comment on the content and modifications using the Discussion tab on the content’s wiki page.

* All ICAR members and the public will be able to view content and add to pages’ Discussion tab. Only Administrators, Chairs and Authors will be able to edit content on specific Sections.

* The Wiki software always tracks all changes made to each page. Changes can be viewed by accessing the History tab on each page.

* The name of the Section page(s) includes the following information:

# The word "Section" and the number of the section of the Guideline followed by a colon (:)
# The name of the Section of the Guideline

Example: Section 5: Conformation recording (the name refers to Section 5 of the Guidelines, related to the Conformation recording )

* Authors are required to follow the instructions for formatting and layout provided in the ICAR Guidelines Wiki SOP, which is accessed on any page via the Navigation menu.

* When a Sub-Committee or Working Group indicates that the final version of a Section or modification is available the Chair will communicate to the Administrator that it is proposed for endorsement by the Board. The Administrator will provide to the Board members the link to the Section or modifications along with instructions. The Board Members should indicate their comments or approval using the Discussion tab on the page being evaluated. Additionally, the Administrator with designate the current draft page(s) as approved (history tab) and place the following note at the top of the proposed draft's page:
<blockquote>
'''NOTE: This version of Section XX 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]].'''
</blockquote>
* Once Board approval is completed the Administrator will send the link for the new content to the ICAR Full Members (cc to Associate members) for comments/approval. No more than two months will be provided for getting back their observations. Their comments/approval should be made in the Discussion tab of the new content’s page. In case of no reply within the two months it is assumed that they agree with the proposed text.

* In consultation with the Chief Executive and the appropriate Chair, the administrator determines if any comment (Discussion tab) requires revisions. In the event revisions are necessary the Administrator will communicate to the appropriate Chair. It is upon the Chair to work with the Authors and Group member to make revisions. Once the revisions are complete the Board, and then the ICAR Members, will be notified by the Administrator and given an opportunity to re-review the content.

* Once all comments have been evaluated and modifications are complete the Administrator marks the final version of the page as APPROVED using the history tab and remove the note about the approval by the chair.

https://www.mediawiki.org/wiki/Extension:Approved_Revs

Approval of Page Process

2025-04-25T14:20:30Z

Bgolden:

[[Category: SOP]]

'''PURPOSE'''

Description of the procedures for production, revision and editing of the ICAR Guidelines Wiki implementation.

'''SCOPE'''

Coordinating the ICAR Team in producing, maintaining and editing of the proper style, and organisation of the ICAR Guidelines Wiki implementation.

'''PROJECT TEAM'''

'''Chief Executive'''

'''Working Groups''': The groups designated to be responsible for drafting, and editing Guidelines Wiki Sections’ content.

'''Sub-Committee''': Any part of a Working Group designated as a content creator for a portion of the Guidelines Wiki Section.

'''Administrator:''' individual(s) whose Wiki account has all management permissions enabled.

'''PROCEDURES & RESPONSIBILITIES'''

* The production of the Guidelines Wiki content is the responsibility of each Working Group and Sub-Committee.

* The Administrator will follow the development of the Guidelines Wiki and the meetings of the Sub-Committee and Working Group for their production and/or editing.

* For equations, the Sub-Committee and Working Group will provide the reference for any future evaluation of the correctness of the equation reported in the Guidelines.

* The Working Group or Sub-Committee Chair will designate at least two members (Authors) to the Administrator. These Authors will be given accounts with permission to modify and add text to only their specific Guidelines Wiki Section. Other members will comment on the content and modifications using the Discussion tab on the content’s wiki page.

* All ICAR members and the public will be able to view content and add to pages’ Discussion tab. Only Administrators, Chairs and Authors will be able to edit content on specific Sections.

* The Wiki software always tracks all changes made to each page. Changes can be viewed by accessing the History tab on each page.

* The name of the Section includes the following information:

# The word "Section" and the number of the section of the Guideline followed by a colon (:)
# The name of the Section of the Guideline

Example: Section 5: Conformation recording (the name refers to the Section 5 of the Guidelines, related to the Conformation recording )

* Authors are required to follow the instructions for formatting and layout provided in the ICAR Guidelines Wiki SOP, which is accessed on any page via the Navigation menu.

* When a Sub-Committee or Working Group indicates that the final version of a Section or modification is available the Chair will communicate to the Administrator that it is proposed for endorsement by the Board. The Administrator will provide to the Board members the link to the Section or modifications along with instructions. The Board Members should indicate their comments or approval using the Discussion tab on the page being evaluated. Additionally, the Administrator with designate the current draft page(s) as approved (history tab) and place the following note at the top of the proposed draft's page:
<blockquote>
'''NOTE: This version of Section XX 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]].'''
</blockquote>
* Once Board approval is completed the Administrator will send the link for the new content to the ICAR Full Members (cc to Associate members) for comments/approval. No more than two months will be provided for getting back their observations. Their comments/approval should be made in the Discussion tab of the new content’s page. In case of no reply within the two months it is assumed that they agree with the proposed text.

* In consultation with the Chief Executive and the appropriate Chair, the administrator determines if any comment (Discussion tab) requires revisions. In the event revisions are necessary the Administrator will communicate to the appropriate Chair. It is upon the Chair to work with the Authors and Group member to make revisions. Once the revisions are complete the Board, and then the ICAR Members, will be notified by the Administrator and given an opportunity to re-review the content.

* Once all comments have been evaluated and modifications are complete the Administrator marks the final version of the page as APPROVED using the history tab and remove the note about the approval by the chair.

https://www.mediawiki.org/wiki/Extension:Approved_Revs

Approval of Page Process

2025-04-25T14:19:03Z

Bgolden:

[[Category: SOP]]

'''PURPOSE'''

Description of the procedures for production, revision and editing of the ICAR Guidelines Wiki implementation.

'''SCOPE'''

Coordinating the ICAR Team in producing, maintaining and editing of the proper style, and organisation of the ICAR Guidelines Wiki implementation.

'''PROJECT TEAM'''

'''Chief Executive'''

'''Working Groups''': The groups designated to be responsible for drafting, and editing Guidelines Wiki Sections’ content.

'''Sub-Committee''': Any part of a Working Group designated as a content creator for a portion of the Guidelines Wiki Section.

'''Administrator:''' individual(s) whose Wiki account has all management permissions enabled.

'''PROCEDURES & RESPONSIBILITIES'''

* The production of the Guidelines Wiki content is the responsibility of each Working Group and Sub-Committee.

* The Administrator will follow the development of the Guidelines Wiki and the meetings of the Sub-Committee and Working Group for their production and/or editing.

* For equations, the Sub-Committee and Working Group will provide the reference for any future evaluation of the correctness of the equation reported in the Guidelines.

* The Working Group or Sub-Committee Chair will designate at least two members (Authors) to the Administrator. These Authors will be given accounts with permission to modify and add text to only their specific Guidelines Wiki Section. Other members will comment on the content and modifications using the Discussion tab on the content’s wiki page.

* All ICAR members and the public will be able to view content and add to pages’ Discussion tab. Only Administrators, Chairs and Authors will be able to edit content on specific Sections.

* The Wiki software always tracks all changes made to each page. Changes can be viewed by accessing the History tab on each page.

* The name of the Section includes the following information:

# The number of the section of the Guideline
# The name of the Section of the Guideline

Example: Section 5: Conformation recording (the name refers to the Section 5 of the Guidelines, related to the Conformation recording )

* Authors are required to follow the instructions for formatting and layout provided in the ICAR Guidelines Wiki SOP, which is accessed on any page via the Navigation menu.

* When a Sub-Committee or Working Group indicates that the final version of a Section or modification is available the Chair will communicate to the Administrator that it is proposed for endorsement by the Board. The Administrator will provide to the Board members the link to the Section or modifications along with instructions. The Board Members should indicate their comments or approval using the Discussion tab on the page being evaluated. Additionally, the Administrator with designate the current draft page(s) as approved (history tab) and place the following note at the top of the proposed draft's page:
<blockquote>
'''NOTE: This version of Section XX 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]].'''
</blockquote>
* Once Board approval is completed the Administrator will send the link for the new content to the ICAR Full Members (cc to Associate members) for comments/approval. No more than two months will be provided for getting back their observations. Their comments/approval should be made in the Discussion tab of the new content’s page. In case of no reply within the two months it is assumed that they agree with the proposed text.

* In consultation with the Chief Executive and the appropriate Chair, the administrator determines if any comment (Discussion tab) requires revisions. In the event revisions are necessary the Administrator will communicate to the appropriate Chair. It is upon the Chair to work with the Authors and Group member to make revisions. Once the revisions are complete the Board, and then the ICAR Members, will be notified by the Administrator and given an opportunity to re-review the content.

* Once all comments have been evaluated and modifications are complete the Administrator marks the final version of the page as APPROVED using the history tab and remove the note about the approval by the chair.

https://www.mediawiki.org/wiki/Extension:Approved_Revs

Section 20: Comparison of methods to measure methane

2025-04-25T14:09:56Z

Bgolden:

Section 20: Pro's and con's of devices

2025-04-25T14:09:31Z

Bgolden: Bgolden moved page Pro's and con's of devices to Section 20: Pro's and con's of devices without leaving a redirect

Section 20: Correlations among methods

2025-04-25T14:08:46Z

Bgolden: Bgolden moved page Correlations among methods to Section 20: Correlations among methods without leaving a redirect

Section 20 – Methane Emission for Genetic Evaluation

2025-04-25T14:07:05Z

Bgolden:

<center>
<big>
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]].

</big>
</center>
== Introduction ==
Increases in milk production through management and genetics have substantially improved feed efficiency and decreased costs per unit of product over recent decades. However, dairy systems are also associated with environmental costs (Baskaran et al., 2009<ref>Baskaran, R., Cullen, R., and Colombo, S. 2009. Estimating values of environmental impacts of dairy farming in New Zealand, New Zealand J. Agric. Res. 52: 377-389, DOI: 10.1080/00288230909510520.</ref>), with methane (CH4) emissions associated with rumen microbial fermentation being both an important contributor to global greenhouse gas (GHG) emissions, as well as an avoidable loss of energy that could otherwise be directed into milk production. The livestock sector is responsible for 14.5% of the global GHG (Gerber et al., 2013<ref>Gerber, P.J., Steinfeld, H., Henderson, B., Mottet, A., Opio, C., Dijkman, J., Falcucci, A. , and Tempio, G. 2013. Tackling climate change through livestock – A global assessment of emissions and mitigation opportunities. Food and Agriculture Organization of the United Nations (FAO), Rome.</ref>); dairy cattle account for 18.9% of these emissions, mainly in the form of enteric CH4 emissions (van Middelaar et al., 2014<ref>Van Middelaar, C.E., Dijkstra. J., Berentsen. P.B.M.. and De Boer. I.J.M. 2014. Cost-effectiveness of feeding strategies to reduce greenhouse gas emissions from dairy farming. J. Dairy Sci. 97:2427–2439.</ref>). Methane is a greenhouse gas with a global warming potential 28 times that of CO2 (Myhre et al., 2013<ref>Myhre, G., Shindell, D., Bréon, F., Collins, W., Fuglestvedt, J., Huang, J., Koch, D., Lamarque, J., Lee, D., Mendoza, B., and Nakajima ,T. 2013. Anthropogenic and Natural Radiative Forcing. T.F. Stocker, D. Qin, G.-K. Plattner, M. Tignor, S.K. Allen, J. Boschung, A. Nauels, Y. Xia, V. Bex, and P.M. Midgley, ed. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA.</ref>). Methane from ruminant livestock is generated during microbial fermentation in the rumen and hindgut (enteric CH4), and from decomposition of manure. Enteric CH4 contributes 80% of CH4 emissions by ruminants, and manure decomposition contributes 20%. Enteric CH4 accounts for 17% of global CH4 emissions and 3.3% of total global greenhouse gas emissions from human activities (Knapp et al., 2014<ref>Knapp, J.R., Laur, G.L., Vadas, P.A., Weis,s W.P., and Tricarico, J.M. 2014. Invited review: Enteric methane in dairy cattle production: Quantifying the opportunities and impact of reducing emissions. J. Dairy Sci. 97:3231-3261.</ref>). There is, therefore, a significant research interest to find ways to reduce enteric CH4 emissions by ruminants. Ruminant animals have a digestive system to digest plant materials efficiently. Like most mammals, ruminants lack the cellulase enzyme required to break the beta-glucose linkages in cellulose, but they play host to diverse populations of rumen microbes that can digest cellulose and other plant constituents. When rumen bacteria, protozoa and fungi ferment carbohydrates and proteins of plant materials, they produce volatile fatty acids, principally acetate, propionate and butyrate. High fibre diets favour acetate synthesis. Synthesis of acetate and butyrate are accompanied by release of metabolic hydrogen, which, if allowed to accumulate in rumen fluid, has negative effects on microbial growth, and feed digestibility (Janssen, 2010<ref>Janssen, P.H. 2010. Influence of hydrogen on rumen methane formation and fermentation balances through microbial growth kinetics and fermentation thermodynamics. Anim. Feed Sci. Technol. 160:1–22. doi:10.1016/j.anifeedsci.2010.07.002.</ref>). Rumen Archaea are microorganisms that combine metabolic hydrogen with CO2 to produce CH4 and water. Archaea play a vital role, therefore, in protecting the rumen from excess metabolic hydrogen, and the CH4 they produce is an inevitable product of rumen fermentation. A number of CH4 phenotypes have been defined (Hellwing et al., 2012<ref>Hellwing, A.L.F., Lund, P., Weisbjerg, M.R., Brask, M., and Hvelplund. T. 2012. Technical note: test of a low-cost and animal-friendly system for measuring methane emissions from dairy cows. J. Dairy Sci. 95:6077–85. doi:10.3168/jds.2012-5505.</ref>); the most widely used is CH4 production (MeP) in liters or grams per day. The CH4 production trait is highly correlated with feed intake (Basarab et al., 2013<ref>Basarab, J., Beauchemin, K., Baron, V., Ominski, K., Guan, L., Miller, S., and Crowley, J. 2013. Reducing GHG emissions through genetic improvement for feed efficiency: Effects on economically important traits and enteric methane production. Animal, 7(S2):303-315. doi:10.1017/S1751731113000888</ref>; De Haas et al., 2017<ref name=":0">de Haas, Y., Pszczola, M., Soyeurt, H., Wall, E., and Lassen, J. 2017. Invited review: Phenotypes to genetically reduce greenhouse gas emissions in dairying. J. Dairy Sci. 100:855-870.</ref>) and, thereby, with the ultimate breeding goal trait: milk production in dairy cattle. The economic value of daily dry matter intake and associated methane emissions in dairy cattle showed that increasing the feed performance estimated breeding value by one unit (i.e. 1 kg of more efficiently converted DMI during the cow’s first lactation) translates to a total lifetime saving of 3.23 kg in DMI and 0.055 kg in methane (Richardson et al., 2019<ref>Richardson, C., Baes, C., Amer, P., Quinton, C., Martin, P., Osborne, V., Pryce, J.E., and Miglior, F. 2020. Determining the economic value of daily dry matter intake and associated methane emissions in dairy cattle. Animal 14:171-179. doi:10.1017/S175173111900154X</ref>). Feed Performance was defined as a 1 kg increase in more efficiently used feed in a first parity lactating cow. These results show not only the relation between DMI and CH4 production, but also the economic relationship between these traits. Persistency of lactation was found to be positively associated with increased feed efficiency and decreased methane production and intensity. Feed efficiency was associated with lower methane intensity. Feed efficiency and methane emissions can be improved by selecting for dairy cattle that are smaller and have increased persistency of lactation. Efficiency and methane emissions can be further improved by improved management of body condition score and by extending lactations beyond the conventional 305-day length (Seymour, 2019<ref>Seymour, D.J. 2019. Feed Efficiency Dynamics in Relation to Lactation and Methane Emissions in Dairy Cattle. PhD thesis, The University of Guelph, Canada.</ref>). According to Ellis et al. (2007)<ref>Ellis, J.L., Kebreab, E., Odongo, N.E., McBride, B.W., Okine, E.K., and France, J. 2007. Prediction of methane production from dairy and beef cattle. J. Dairy Sci. 90:3456–3466.</ref>, DMI predicted MeP with an R2 of 0.64, and ME intake (MJ/d) predicted MeP with an R2 of 0.53 for dairy cattle. AlternativePhenotype definitions include CH4 intensity (MeI), which is defined as liters or grams of CH4 per kg of milk, and CH4 yield (MeY), which is defined as liters or grams of CH4 per kg of dry matter intake (DMI) (Moate et al., 2016<ref>Moate, P.J., Deighton, M.H., Williams, S.R.O., Pryce, J.E., Hayes, B.J., Jacobs, J.L., Eckard, R.J., Hannah, M.C. and Wales, W.J., 2016. Reducing the carbon footprint of Australian milk production by mitigation of enteric methane emissions. Anim. Prod. Sci. 56:1017-1034.</ref>). Residual CH4 production (RMP) is calculated as observed minus predicted CH4 production (Herd et al., 2014<ref>Herd, R.M., Arthur, P.F., Bird, S.H., Donoghue, K.A., and Hegarty, R.S. 2014. Genetic variation for methane traits in beef cattle. In: Proc. 10th World Conference on Genetic Applied to Livestock Production (WCGALP), 17-22 August, 2014. Vancouver, Canada.</ref>, Berry et al., 2015<ref>Berry, D.P., Lassen, J., and de Haas, Y. 2015. Residual feed intake and breeding approaches for enteric methane mitigation In: Livestock production and climate change. P.K. Malik, R. Bhatta, J. Takahashi, R.A. Kohn, and C.S. Prasad, ed. CABI, Oxfordshire, UK. . Pages 273-291</ref>), with predicted values based on factors such as milk production, body weight and feed intake. At the moment, it is not obvious which of these phenotypes to use; but, it is important to monitor associations between the chosen CH4 phenotype and the other important traits in the breeding goal (e.g. production, fertility, longevity) to avoid unfavorable consequences. Berry and Crowley (2012)<ref>Berry, D.P., and Crowley, J.J. 2012. Residual intake and body weight gain: A new measure of efficiency in growing cattle, J. Anim. Sci. 90:109–115, <nowiki>https://doi.org/10.2527/jas.2011-4245</nowiki></ref> describe advantages and limitations of ration traits. For example, because feed efficiency traits are a linear combination of other traits it is not recommended to include them in an overall total merit index, which is a clear limitation. For all applications it is necessary to measure the CH4 emission of each animal individually. These guidelines are intended to make the right choices for this. Whilst diet changes and feed additives can be effective mitigation strategies for CH4 emissions (Beauchemin et al., 2009<ref>Beauchemin, K.A., McAllister, T.A., and McGinn, S.M. 2009. Dietary mitigation of enteric methane from cattle. CAB Rev.: Perspect. Agric., Vet. Sci., Nutr. Nat. Res. 4:1–18.</ref>; Martin et al., 2010<ref>Martin C., Morgavi, D.P., and Doreau, M. 2010. Methane mitigation in ruminants: from microbe to the farm scale. Animal 4:351–365.</ref>; Hristov et al., 2013<ref>Hristov, A.N., Oh, J., Firkins, J.L., Dijkstra, J., Kebreab, E., Waghorn, G., Makkar, H.P.S., Adesogan, A.T., Yan,g W., Lee, C., Gerber, P.J., Henderson, B., and Tricarico, J.M. 2013. Special topics - Mitigation of methane and nitrous oxide emissions from animal operations: I. A review of enteric methane mitigation options. J. Anim. Sci. 91:5045–5069.</ref>), their effects depend on the continued use of a particular diet or additive and there have been issues with the rumen microbiomes adapting to additives. Rumen bacterial communities are highly dynamic after a diet switch and did not stabilize within 5 wk of cows grazing pasture (Bainbridge et al., 2016<ref>Bainbridge, M.L., Saldinger, L.K., Barlow, J.W., Alvez, J.P., Roman, J. Kraft, J. 2016. 1609 Rumen bacterial communities continue to shift five weeks after switching diets from conserved forage to pasture. J. Anim. Sci. 94, suppl_5:783, <nowiki>https://doi.org/10.2527/jam2016-1609</nowiki> (abstr.)</ref>). In contrast, breeding for reduced CH4 emissions should result in a permanent and cumulative reduction of emissions (Wall et al., 2010<ref>Wall, E., Simm, G., and Moran, D. 2010. Developing breeding schemes to assist mitigation of greenhouse gas emissions. Animal 4:366-376.</ref>). Several studies have shown that CH4 emissions by ruminants have a genetic component, with heritability in the range 0.20 – 0.30 (de Haas et al., 2011<ref>de Haas, Y., Windig, J.J., Calus, M.P.L., Dijkstra, J., de Haan, M., Bannink, A., and Veerkamp, R F. 2011. Genetic parameters for predicted methane production and potential for reducing enteric emissions through genomic selection. J. Dairy Sci. 94:6122–6134.</ref>; Donoghue et al., 2013<ref>Donoghue K.A., Herd, R.M., Bird, S.H., Arthur, P.F., and Hegarty, R F. 2013. Preliminary genetic parameters for methane production in Australian beef cattle. In: Proceedings of the Association for the Advancement of Animal Breeding and Genetics, 20-23 October 2013, Napier, New Zealand, pp. 290–293.</ref>; Pinares-Patiño et al., 2013<ref>Pinares-Patiño C.S., Hickey, S.M., Young, E.A., Dodds, K.G., MacLean, S., Molano, G., Sandoval, E., Kjestrup, H., Harland, R., Pickering, N.K., and McEwan, J.C. 2013. Heritability estimates of methane emissions from sheep. Animal 7: 316–321.</ref>, Kandel et al., 2014A<ref>Kandel, P.B., Vanderick, S., Vanrobays, M.L., Vanlierde, A., Dehareng, F., Froidmont, E., Soyeurt, H., and Gengler, N. 2014A. Consequences of selection for environmental impact traits in dairy cows. Page 19. (<nowiki>http://orbi.ulg.ac.be/bitstream/2268/164402/164401/NSABS162014_poster_Purna_abstract.pdf</nowiki>) I:n Proc. 19th National symposium on applied biological sciences, Gembloux, Belgium.</ref>, B<ref>Kandel, P.B., Vanderick, S., Vanrobays, M.L., Vanlierde, A., Dehareng, F., Froidmont, E., Soyeur,t H., and Gengler, N. 2014B. Consequences of selection for environmental impact traits in dairy cows. In: 10th World Congress on Genetics Applied to Livestock Production (WCGALP), 17-22 August, 2014. Vancouver, Canada.</ref>; Lassen and Lovendahl, 2016<ref>Lassen, J., and Løvendahl, P. 2016. Heritability estimates for enteric methane emissions from Holstein cattle measured using noninvasive methods. J. Dairy Sci. 99:1959-1967.</ref>; López-Paredes et al. 2020<ref>Lopez-Paredes, J., Goiri, I., Atxaerandio, R., García-Rodríguez, A., Ugarte, E., Jiménez-Montero, J.A., Alenda, R and González-Recio, O. 2020. Mitigation of greenhouse gases in dairy cattle via genetic selection (i): Genetic parameters of direct methane using non-invasive methods and its proxies. J. Dairy Sci. 103.</ref>). Breeding for reduced CH4 emissions, alone or together with other mitigation strategies, could therefore be effective in reducing the environmental impact of cattle farming and, possibly, also in increasing feed efficiency. Such a breeding scheme would require, as a fundamental starting point, accurate measures of individual CH4 emissions on a large scale. Several techniques have been developed for the measurement of CH4 emissions from ruminants, with varying degrees of accuracy (see reviews by Cassandro et al., 2013<ref>Cassandro, M., Mele, M., Stefanon, B.. 2013. Genetic aspects of enteric methane emission in livestock ruminants. Italian J. Anim. Sci. 12:e73: 450-458.</ref> and Hammond et al., 2016A<ref>Hammond, K.J., Crompton, L.A., Bannink, A., Dijkstra, J., Yáñez-Ruiz, D.R., O’Kiely, P., Kebreab, E., Eugenè, M.A., Yu, Z., Shingfield, K.J., Schwarm, A., Hristov, A.N., and Reynolds, C.K. 2016A. Review of current in vivo measurement techniques for quantifying enteric methane emission from ruminants. Anim. Feed Sci. Technol. 219:13–30. doi:10.1016/j.anifeedsci.2016.05.018.</ref>), but routine individual measurements on a large scale (a requisite for genetic selection) have proven to be difficult to obtain and expensive to measure (Pickering et al., 2015<ref>Pickering, N.K., Oddy, V.H., Basarab, J.A., Cammack, K., Hayes, B J., Hegarty, R.S., McEwan, J.C., Miller, S., Pinares, C., and de Haas, Y. 2015. Invited review: Genetic possibilities to reduce enteric methane emissions from ruminants. Animal 9:1431-1440.</ref>; Negussie et al., 2016<ref>Negussie E., Lehtinen, J., Mäntysaari, P., Liinamo, A-E., Mäntysaari, E., and Lidauer, M.. 2016. Non-invasive individual methane measurements in dairy cows using photoacoustic infrared spectroscopy technique. 6th Greenhouse Gases Animal Agriculture Conference (GGAA2016) 14-18 February 2016. Melbourne, Australia. Abstract. p62.</ref>). Therefore, identifying proxies (i.e. indicators or indirect traits) that are correlated to CH4 emissions, but which are easy and relatively lowcost to record on a large scale, would be a welcome alternative. Proxies might be less accurate but could be measured repeatedly to reduce random noise and in much larger populations. These guidelines are highly indebted to Garnsworthy et al. (2019)<ref name=":1">Garnsworthy, P.C. Difford, G.F. Bell, M.J. Bayat, A.R. Huhtanen, P. Kuhla, B. Lassen, J. Peiren, N. Pszczola, M; Sorg, D. Visker, M.H., and Yan, T. 2019 Comparison of Methods to Measure Methane for Use in Genetic Evaluation of Dairy Cattle. Animals 9:837, 12p.</ref>. In this paper the methods to measure CH4 are compared with special emphasis to the genetic evaluation of dairy cattle.

== Disclaimer ==
The fact that specific device manufacturers are mentioned in these guidelines is in no way an endorsement of the devices or their accuracy by ICAR.

== Scope ==
A variety of technologies are being developed and employed to measure CH4 emissions of individual dairy cattle under various environmental conditions, as is evidenced by frequent reviews (Storm et al., 2012<ref>Storm, I.M., Hellwing, A.L.F., Nielsen, N.I., and Madsen, J. 2012. Methods for measuring and estimating methane emission from ruminants. Animals 2:160-183.</ref>; Cassandro et al., 2013<ref>Cassandro, M., Mele, M., Stefanon, B.. 2013. Genetic aspects of enteric methane emission in livestock ruminants. Italian J. Anim. Sci. 12:e73: 450-458.</ref>; Hammond et al., 2016A<ref>Hammond, K.J., Crompton, L.A., Bannink, A., Dijkstra, J., Yáñez-Ruiz, D.R., O’Kiely, P., Kebreab, E., Eugenè, M.A., Yu, Z., Shingfield, K.J., Schwarm, A., Hristov, A.N., and Reynolds, C.K. 2016A. Review of current in vivo measurement techniques for quantifying enteric methane emission from ruminants. Anim. Feed Sci. Technol. 219:13–30. doi:10.1016/j.anifeedsci.2016.05.018.</ref>; de Haas et al., 2017<ref>de Haas, Y., Pszczola, M., Soyeurt, H., Wall, E., and Lassen, J. 2017. Invited review: Phenotypes to genetically reduce greenhouse gas emissions in dairying. J. Dairy Sci. 100:855-870.</ref>). The first objective of the current guidelines is to review and compare the suitability of methods for large-scale measurements of CH4 output of individual animals, which may be combined with other databases for genetic evaluations. Comparisons include assessing the accuracy, precision and correlation between methods. Combining datasets from different countries and research centres could be a successful strategy for making genetic progress in this difficult to measure trait if the methods are correlated (de Haas et al., 2017<ref name=":0" />). Accuracy and precision of methods are important. Data from different sources need to be appropriately weighted or adjusted when combined, so any methods can be combined if they are suitably correlated with the ‘true’ value. The second objective of the current guidelines, therefore, is to examine correlations among results obtained by different methods, ultimately leading to an estimate of confidence limits for selecting individual animals that are high or low emitters (see also Garnsworthy et al., 2019<ref name=":1" />).

== Sub-sections ==
<div style="column-count:2">
:[[Section 20: Definition and Terminology | Definition and Terminology]]
:[[Section 20: Methane determining factors | Methane determining factors]]
:[[Section 20: Methane measuring methods|Methane measurements methods]]
:[[Section 20: Discussion of methods | Discussion of methods]]
:[[Section 20: Comparison of methods to measure methane | Comparison of methods to measure methane]]
:[[Section 20: Proxies | Proxies]]
:[[Section 20: Proxies Discussion|Proxies discussion]]
:[[Section 20: Merging and sharing data in genetic evaluations | Merging and sharing data in genetic evaluations]]
:[[Section 20: Ongoing activities | Ongoing activities]]
:[[Section 20: Recommendations|Recomendations]]
:[[Section 20: Conclusions | Conclusions]]
</div>

== Summary of Changes ==
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|March 2020
|Draft from Feed & Gas WG put into standard template for ICAR Guidelines. Separate out EDGP database to become a standalone appendix.
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|April 2020
|Edits and acknowledgements added by Feed & Gas WG.
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|May 2020
|Approved by ICAR Board on 26th May subject to addition of disclaimer.
Disclaimer added as new chapter 2 - the fact specific device manufacturers are mentioned in these guidelines is in no way an endrosement of the devices or their accuracy by ICAR.
|-
|December 2023
|Creation of Methane Emission for Genetic Evaluation Wiki Page.
|}
==References==

Section 20: Definition and Terminology

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Section 20: Methane determining factors

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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]].

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== Diet and rumen microbiota ==
Table 1 contains a list of dietary or microbiota factors that determine CH4 production.
{| class="wikitable"
|+
!Factors
!Reference
|-
|The main determinants of daily methane production are dry matter intake and diet composition: the more feed consumed, and/or the greater the fibre content of the diet, the more methane is produced per day. However, per unit of DMI, and per unit of fat+protein yield the grass diet produced less enteric CH4 per cow than the TMR diet. Nutritional approaches for methane mitigation include reducing the forage to concentrate ratio of diets, increasing dietary oil content, and dietary inclusion of rumen modifiers and methane inhibitors.
|Beauchemin et al., 2009<ref>Beauchemin, K.A., McAllister, T.A., and McGinn, S.M. 2009. Dietary mitigation of enteric methane from cattle. CAB Rev.: Perspect. Agric., Vet. Sci., Nutr. Nat. Res. 4:1–18.</ref>;
Cottle et al., 2011<ref>Cottle, D.J., Nolan, J.V., and Wiedemann, S.G. 2011. Ruminant enteric methane mitigation: A review. Anim. Prod. Sci. 51:491–514. doi:10.1071/AN10163.</ref>; Knapp et al., 2014<ref>Knapp, J.R., Laur, G.L., Vadas, P.A., Weis,s W.P., and Tricarico, J.M. 2014. Invited review: Enteric methane in dairy cattle production: Quantifying the opportunities and impact of reducing emissions. J. Dairy Sci. 97:3231-3261.</ref>; O’Neill et al., 2011<ref>O’Neill, B.F., Deighton, M.H., O’Loughlin, B.M., Mulligan, F.J., Boland, T.M., O’Donovan, M., and Lewis, E. 2011. Effects of a perennial ryegrass diet or total mixed ration diet offered to spring-calving Holstein-Friesian dairy cows on methane emissions, dry matter intake, and milk production. J. Dairy Sci. 94:1941 – 1951</ref>; Sauvant et al., 2011<ref>Sauvant, D., Giger-Reverdin, S., Serment, A., and Broudiscou, L. 2011. Influences des régimeset de leur fermentation dans le rumen sur la production de méthane par les ruminants. INRA Prod. Anim. 24:433–446.</ref>
|-
|Methane output per kg of product is affected mainly by cow milk yield or growth rate, and by herd-level factors, such as fertility, disease incidence and replacement rate.
|Garnsworthy, 2004<ref>Garnsworthy, P.C. 2004. The environmental impact of fertility in dairy cows: a modelling approach to predict methane and ammonia emissions. Anim. Feed Sci. Technol. 112:211-223.</ref>
|-
|Methane output varies considerably between individual animals. For animals fed the same feed, the between-animal coefficient of variation (CV) in methane was 8.1%.
|Blaxter and Clapperton, 1965<ref>Blaxter, K.L., and Clapperton, J.L. 1965. Prediction of the amount of methane produced by ruminants. Br. J. Nutr.19:511–522.</ref>
|-
|The amount of digestible nutrients consumed especially of the carbohydrate fraction (starch, sugar, N-free residuals) is reliable to estimate CH4 release with high precision. Furthermore, diets rich in fat reduced CH4 formation in the rumen.
|Jentsch et al., 2007<ref>Jentsch, W., Schweigel, M., Weissbach, F., Scholze, H., Pitroff, W., and Derno, M. 2007. Methane production in cattle calculated by the nutrient composition of the diet. Arch. Anim. Nutr. 61:10-19.</ref>
|-
|DMI was also the most important determining factor, but there were different regression lines for maize silage and dried grass as the main roughage component respectively:
<math>CH4\ (g)=93+16.8\times DMI(kg)</math>
<math>CH4\ (g)=81+14.0\times DMI(kg)</math>

Methane release was particularly dependent on the intake of crude fiber (CF) and ether extract (EE):

<math>CH4\ (g)=63+80xCF\ (kg)+11xNFE\ (kg)+19xCP(kg)-195xEE\ (kg)</math>
|Kirchgessner et al., 1991<ref>Kirchgessner, M., Windisch, W., Müller, H. L., and Kreuzer, M. 1991. Release of methane and of carbon dioxide by dairy cattle. Agribiol. Res. 44:91-102.</ref>
|-
|Methane linearly increased with NDF intake <math>CH4\ (L)=59.4\times NDF[kg]+ 64.6</math> for cows together with their calves independent of the breed.
|Estermann et al., 2002<ref>Estermann, B.L., Sutter, F., Schlegel, P.O., Erdin, D., Wettstein, H.R., and Kreuzer, M. 2002. Effect of calf age and dam breed on intake, energy expenditure, and excretion of nitrogen, phosphorus, and methane of beef cows with calves. J. Anim. Sci. 80:1124-1134.</ref>
|-
|Enteric CH4 could be predicted with the equation:
<math>CH4\ (g/d)=84+47\times cellulose(kg/d)+32\times starch(kg/d)+62\times sugars\ (kg/d)</math>
|Hindrichsen et al., 2005<ref>Hindrichsen, I.K., Wettstein, H.R., Machmüller, A., Jörg, B., and Kreuzer, M. 2005. Effect of the carbohydrate composition of feed concentratates on methane emission from dairy cows and their slurry. Environ. Monit. Assess., 107:329-350.</ref>
|-
|The higher the percentage concentrate the lower Ym.
|Zeitz et al., 2012<ref>Zeitz, J.O., Soliva, C.R., and Kreuzer, M. 2012. Swiss diet types for cattle: how accurately are they reflected by the Intergovernmental Panel on Climate Change default values? J. Int. Environ. Sci. 9(sup1):199-216.</ref>
|-
|Additives can sometimes have a methane reducing effect: higher dosages mitigate methane more. Saponins mitigate methanogenesis by reducing the number of protozoa, whereas condensed tannins act both by reducing the number of protozoa and by a direct toxic effect on methanogens.
|Beauchemin et al., 2008;<ref>Beauchemin, K.A., Kreuze,r M., O’Mara, F., and McAllister, T.A. 2008. Nutritional management for enteric methane abatement: A review. Aust. J. Exp. Agric. 48:21–27.</ref>
Jayanegara et al.<ref>Jayanegara, A., Leiber, F., and Kreuzer, M. 2012. Meta‐analysis of the relationship between dietary tannin level and methane formation in ruminants from in vivo and in vitro experiments. J. Anim. Physiol. Anim. Nutr. 96:365-375.</ref>, 2012; Zmora et al., 2012<ref>Zmora, P., Cieslak, A., Pers-Kamczyc, E., Nowak, A., Szczechowiak, J. and Szumacher-Strabel, M. 2012. Effect of Mentha piperita L. on in vitro rumen methanogenesis and fermentation, Acta Agr. Scan. Section A — Anim. Sci. 62:46-52, DOI: 10.1080/09064702.2012.703228.</ref>; Cieslak et al., 2013<ref>Cieslak, A., Szumacher-Strabel, M., Stochmal, A., nad Oleszek, W. 2013. Plant components with specific activities against rumen methanogens. Animal, 7(s2):253-265.</ref>; Guyader et al., 2014<ref>Guyader, J., Eugène, M., Nozière, P., Morgavi, D.P., Doreau, M., and Martin, C. 2014. Influence of rumen protozoa on methane emission in ruminants: a meta-analysis approach. Animal 8:1816-1825.</ref>
|-
|Plant essential oils have been shown as promising feed additives to mitigate CH4 and ammonia emission, but results were inconsistent.
|Cobellis et al., 2016;<ref>Cobellis, G., Trabalza-Marinucci, M. and Yu, Z. 2016. Critical evaluation of essential oils as rumen modifiers in ruminant nutrition: A review. Sci. Total Environ. 545: 556-568.</ref>
Moate et al., 2011<ref>Moate, P.J., Deighton, M.H., Williams, S.R.O., Pryce, J.E., Hayes, B.J., Jacobs, J.L., Eckard, R.J., Hannah, M.C. and Wales, W.J., 2016. Reducing the carbon footprint of Australian milk production by mitigation of enteric methane emissions. Anim. Prod. Sci. 56:1017-1034.</ref>
|-
|Nitrate and sulphate addition decreased the enteric methane emissions negatively affecting diet digestibility and milk production. The effects of the salts are additive.
|van Zijderveld et al., 2010<ref>Van Zijderveld, S.M., Gerrits, W.J.J., Apajalahti, J.A., Newbold, J.R., Dijkstra, J., Leng, R A., and Perdok, H.B. 2010. Nitrate and sulfate: effective alternative hydrogen sinks for mitigation of ruminal methane production in sheep. J. Dairy Sci. 93:5856-5866.</ref>;
van Zijderveld et al., 2011<ref>Van Zijderveld, S.M., Gerrits, W.J.J., Dijkstra, J., Newbold, J.R., Hulshof, R.B.A., and Perdok, H.B. 2011. Persistency of methane mitigation by dietary nitrate supplementation in dairy cows. J. Dairy Sci. 94:4028-4038.</ref>
|-
|The methanogenesis in the rumen of calves is associated with the development of the ruminal protozoa population. The absence of protozoa in the rumen reduced both the CH4 production and the digestibility of carbohydrates.
|Schönhusen et al., 2003<ref>Schönhusen, U., Zitnan, R., Kuhla, S., Jentsch, W., Derno, M., and Voigt, J. 2003. Effects of protozoa on methane production in rumen and hindgut of calves around time of weaning. Arch. Anim. Nutr. 57:279-295.</ref>
|-
|Implementing good grazing management reduced gross energy intake loss as CH4 by 14%.
|Wims et al., 2010<ref>Wims, C.M., Deighton, M.H., Lewis, E., O’Loughlin, B., Delaby, L., Boland, T.M., and O’Donovan, M. 2010. Effect of pregrazing herbage mass on methane production, dry matter intake, and milk production of grazing dairy cows during the mid-season period. J. Dairy Sci. 93:4976 – 4985</ref>
|}Table 1. Methane determining factors related to diet and rumen microbiota.

== Host genetics, physiology and environment ==
A low-moderate proportion of variation in CH4 emissions among ruminants is under genetic control. Heritability coefficients of MeY and RMPR were h²=0.22 and 0.19 respectively in a population of 1,043 Angus growing steers and heifers measured during 2 days in RC (Donoghue et al., 2016<ref>Donoghue, K.A., Bird-Gardiner, T., Arthur, P.F., Herd, R.M., and Hegarty, R.F. 2016. Genetic and phenotypic variance and covariance components for methane emission and postweaning traits in Angus cattle. J. Anim. Sci. 94:1438–1445. doi:10.2527/jas2015-0065.</ref>). The heritability coefficient of MeY was h²=0.13 in a population of1,225 dual-purpose growing sheep measured during 2 days in RC (Pinares-Patino et al., 2013<ref>Pinares-Patiño C.S., Hickey, S.M., Young, E.A., Dodds, K.G., MacLean, S., Molano, G., Sandoval, E., Kjestrup, H., Harland, R., Pickering, N.K., and McEwan, J.C. 2013. Heritability estimates of methane emissions from sheep. Animal 7: 316–321.</ref>). Table 2 contains information of heritability of traits related to CH4 production.
{| class="wikitable"
|+
!Factors
!Reference
|-
|List with several h2
|Pickering et al., 2015<ref>Pickering, N.K., Oddy, V.H., Basarab, J.A., Cammack, K., Hayes, B J., Hegarty, R.S., McEwan, J.C., Miller, S., Pinares, C., and de Haas, Y. 2015. Invited review: Genetic possibilities to reduce enteric methane emissions from ruminants. Animal 9:1431-1440.</ref>
|-
|List with several h2
|MPWG White paper Dec 18<ref>MPWG White paper Dec 18. <nowiki>http://www.asggn.org/publications,listing,95,mpwg-white-paper.html</nowiki>. Pickering, N.K., de Haas, Y., Basarab, J., Cammack, K., Hayes, B., Hegarty, R.S., Lassen, J., McEwan, J.C., Miller, S., Pinares-Patiño, C.S., Shackell, G., Vercoe, P. and Oddy, V.H. 2013.</ref>
|-
|Methane emissions from individual cows during milking varied between individuals with the same milk yield and fed the same diet. Between-cow variation in MERm is greater than within-cow variation and ranking of cows for CH4 emissions is consistent across time. Variation related to body weight, milk yield, parity, and week of lactation/days in milk. The monitored variation might offer opportunities for genetic selection.
|Garnsworthy et al., 2011A<ref>Garnsworthy, P.C., Craigon, J., Hernandez-Medrano, J.H. and Saunders, H. 2012A. On-farm methane measurements during milking correlate with total methane production by individual dairy cows. J. Dairy Sci. 95:3166-3180.</ref>;
Garnsworthy et al., 2011B<ref>Garnsworthy, P.C., Craigon, J., Hernandez-Medrano, J.H., and Saunders, N. 2012B. Variation among individual dairy cows in methane measurements made on farm during milking. J. Dairy Sci. 95:3181–3189.</ref>
|-
|Mechanistic modelling approach: potential for dietary intervention as a means of substantially reducing CH4 emissions without adverse effects on dietary energy supply.
|Mills et al., 2001<ref>Mills, J.A.N., Dijkstra, J., Bannink, A., Cammell, S.B., Kebreab, E., and France, J. 2001. A mechanistic model of whole-tract digestion and methanogenesis in the lactating dairy cow: model development, evaluation, and application. J. Anim. Sci. 79:1584-1597.</ref>
|-
|The CH4-to-CO2 ratio measured using the non-invasive portable air sampler and analyzer unit based on Fourier transform infrared (FTIR) detection method is an asset of the individual cow and may be useful in both management and genetic evaluations.
|Lassen et al., 2012<ref>Lassen, J., Lovendahl, P., and Madsen, J. 2012. Accuracy of noninvasive breath methane measurements using Fourier transform infrared methods on individual cows. J. Dairy Sci. 95:890-898.</ref>
|-
|The estimated heritability for CH4 g/day and CH4 g/kg of FPCM were lower than common production traits but would still be useful in breeding programs.
|Kandel et al., 2013<ref>Kandel, P.B., Vanrobays, M.L., Vanlierde, A., Dehareng, F., Froidmont, E., Dardenne, P., Lewis, E., Buckley, F., Deighton, M.H., McParland, S. and Gengler, N., 2013. Genetic parameters for methane emissions predicted from milk mid-infrared spectra in dairy cows. J. Dairy Sci. 95(E-1):p.388.</ref>
|-
|Genetic correlation between CH4 intensity and milk yield (MY) was - 0.67 and with milk protein yield (PY) was -0.46 in Holstein cows.
|Kandel et al., 2014A, B<ref>Kandel, P.B., Vanderick, S., Vanrobays, M.L., Vanlierde, A., Dehareng, F., Froidmont, E., Soyeur,t H., and Gengler, N. 2014B. Consequences of selection for environmental impact traits in dairy cows. In: 10th World Congress on Genetics Applied to Livestock Production (WCGALP), 17-22 August, 2014. Vancouver, Canada.</ref>
|-
|Milk production and CH4 emissions of dairy cows seemed to be influenced by the temperature humidity index.
|Vanrobays et al., 2013A<ref>Vanrobays, M.-L., Gengler, N., Kandel, P.B., Soyeurt, H., and Hammami, H. 2013A. Genetic effects of heat stress on milk yield and MIR predicted methane emissions of Holstein cows. 64th Annual meeting of the European Federation of Animal Science, p498</ref>
|-
|Estimate the heritability of the estimated methane emissions from 485 Polish Holstein-Friesian dairy cows at 2 commercial farms using FTIR spectroscopy during milking in an automated milking system by implementing the random regression method. The heritability level fluctuated over the course of lactation, starting at 0.23 (SE 0.12) and then increasing to its maximum value of 0.3 (SE 0.08) at 212 DIM and ending at the level of 0.27 ± 0.12. Average heritability was 0.27 ± 0.09.
|Pszczola et al., 2017<ref>Pszczola, M., Rzewuska, K., Mucha, S., and Strabel, T. 2017. Heritability of methane emissions from dairy cows over a lactation measured on commercial farms. J. Anim. Sci. 95:4813-4819. doi: 10.2527/jas2017.1842.</ref>
|-
|CH4 measured with a portable air-sampler FTIR detection method on 3,121 Holstein dairy cows from 20 herds using automatic milking systems. The heritability of CH4_MILK was 0.21 ± 0.06. It was concluded that a high genetic potential for milk production will also mean a high genetic potential for CH4 production. The results suggested that CH4 emission is partly under genetic control, that it is possible to decrease CH4 emission from dairy cattle through selection, and that selection for higher milk yield will lead to higher genetic merit for CH4 emission/cow per day.
|Lassen and Løvendahl, 2016<ref>Lassen, J., and Løvendahl, P. 2016. Heritability estimates for enteric methane emissions from Holstein cattle measured using noninvasive methods. J. Dairy Sci. 99:1959-1967.</ref>
|-
|CH4 production was measured of 184 Holstein-Friesian cows in. the milking robot with a in total 2,456 observations for CH4 production. Heritability for CH4 production ranged from 0.12 ± 0.16 to 0.45 ± 0.11, and genetic correlations with MY ranged from 0.49 ± 0.12 to 0.54 ± 0.26. The positive genetic correlation between CH4 production and milk yield indicates that care needs to be taken when genetically selecting for lower CH4 production, to avoid a decrease in MY at the animal level. However, this study shows that CH4 production is moderately heritable and therefore progress through genetic selection is possible.
|Breider et al., 2019<ref>Breider, I.S., Wall, E., Garnsworthy, P.C. 2019. Short communication: Heritability of methane production and genetic correlations with milk yield and body weight in Holstein-Friesian dairy cows, J. Dairy Sci. 102: 7277-7281.</ref>
|-
|CH4 concentration was measured with NDIR, and CH4 production was estimated from CH4 concentration and body weight. Heritability for CH4 concentration was 0.11 ± 0.03 and for CH4 production 0.12 ± 0.04. Positive genetic correlation was observed with MY (0.17-0.21), PY (0.22-0.31) and FY (0.27-0.29). Other type traits showed positive correlation with methane production (chest width=0.26, angularity =0.19, stature = 0.43 and capacity = 0.31) possibly associated to higher milk feed intake from these animals. Rumination time was negatively correlated to CH4 production (-0.24) and CH4 concentration (-0.43). However, larger CH4 production and CH4 concentration was associated with shorter days open.
|López-Paredes et al. (2020)<ref>Lopez-Paredes, J., Goiri, I., Atxaerandio, R., García-Rodríguez, A., Ugarte, E., Jiménez-Montero, J.A., Alenda, R and González-Recio, O. 2020. Mitigation of greenhouse gases in dairy cattle via genetic selection (i): Genetic parameters of direct methane using non-invasive methods and its proxies. J. Dairy Sci. 103.</ref>
|-
|Genetic parameters of CH4 emissions predicted from milk fatty acid profile (FA) and those of their predictors in 1,091 Brown Swiss cows reared on 85 farms showed that enteric CH4 emissions of dairy cows can be estimated on the basis of milk fatty acid profile. Additive genetic variation of CH4 traits was shown which could be exploited in breeding programmes.
|Bittante and Cecchinato, 2020<ref>Bittante, G., and Cecchinato, A. 2020. Heritability estimates of enteric methane emissions predicted from fatty acid profiles, and their relationships with milk composition, cheese-yield and body size and condition, It. J. An. Sci. 19:114-126, DOI: 10.1080/1828051X.2019.1698979</ref>
|-
|A total of 670 test day records were recorded on lactating Holstein Friesian cows reared in 10 commercial dairy herds. Predicted methane production (PMP) was estimated to be 15.33±1.52 MJ/d in dairy cows with 23.53±6.81 kg/d of milk yeild (MY) and 3.57±0.68% of fat content (FC). Heritability of MY was 0.09 with a posterior probability for values of h2 greater than 0.10 of 44%. Estimates of heritability for FC and protein content (PC) were 0.17 and 0.34, respectively, with a posterior probability for values of h2 greater than 0.10 of 77% and 99%. For somatic cell score (SCS), heritability was 0.13 with a posterior probability for values of h2 greater than 0.10 of 67%. Heritability for the trait PMP was moderate to low (0.12); however, posterior probability for values of h2 greater than 0.10 was 60%. Medians of the posterior distributions of genetic correlations between PMP and milk production traits were: 0.92, 0.67, 0.14, and 0.14 between PMP and MY, PMP and FC, PMP and PC, and PMP and SCS, respectively. Reduction of PMP seems to be viable through selection strategies without affecting udder health and PC.
|Cassandro et al., 2010<ref>Cassandro, M., Cecchinato, A., Battagin, M., Penasa, M., 2010. Genetic parameters of predicted methane production in Holstein Friesian cowsIn: Proc. 9th World Congr. on Genetics Applied to Livestock Production, Leipzig, Germany. . Page 181</ref>
|-
|GWAS to study the genetic architecture of CH4 production and detected genomic regions affecting CH4 production. Detected regions explained only a small proportion of the heritable variance. Potential QTL regions affecting CH4 production were located within QTLs related to feed efficiency, milk-related traits, body size and health status. Five candidate genes were found: CYP51A1 on BTA 4, PPP1R16B on BTA 13, and NTHL1, TSC2, and PKD1 on BTA 25. These candidate genes were involved in a number of metabolic processes that are possibly related to CH4 production. One of the most promising candidate genes (PKD1) was related to the development of the digestive tract. The results indicate that CH4 production is a highly polygenic trait.
|Pszczola et al., 2018<ref>Pszczola, M., Strabel, T., Mucha, S., and Sell-Kubiak, E. 2018. Genome-wide association identifies methane production level relation to genetic control of digestive tract development in dairy cows. Scientific Rep. 8 (1), 15164 <nowiki>https://doi.org/10.1038/s41598-018-33327-9</nowiki></ref>
|-
|A 1000-cow study across European countries revealed that the ruminant microbiomes can be controlled by the host animal. A 39- member subset of the core microbiome formed hubs in co-occurrence networks linking microbiome structure to host genetics and phenotype (CH4 emissions, rumen and blood metabolites, and milk production efficiency).
|Wallace et al.,
2019<ref>Wallace, R.J., Sasson, G., Garnsworthy, P.C., Tapio, I., Gregson, E., Bani, P., Huhtanen, P., Bayat, A.R., Strozzi, F., Biscarini, F., Snelling, T.J., Saunders, N., Potterton, S.L., Craigon, J., Minuti, A., Trevisi, E., Callegari, M.L., Cappelli, F.P., Cabezas-Garcia, E.H., Vilkki, J., Pinares-Patino, C., Fliegerov, K.O., Mrazek, J., Sechovcova, H., Kope, J., Bonin, A., Boyer, F., Taberlet, P., Kokou, F., Halperin, E., Williams, J.L., Shingfield, K.J., and Mizrahi, I. 2019. A heritable subset of the core rumen microbiome dictates dairy cow productivity and emissions. Sci. Adv. 5:(7):eaav8391. doi 10.1126/sciadv.aav8391.</ref>
|}Table 2. Heritability information of methane-related traits and measurements.

Section 20: Methane measuring methods

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Section 20: Discussion of methods

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Section 20: Comparison of methods to measure methane

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Section 20: Proxies

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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]].

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== Introduction ==
Large-scale measurements of enteric CH4 emissions from dairy cows are needed for effective monitoring of strategies to reduce the carbon footprint of milk production, as well as for incorporation of CH4 emissions into breeding programs. However, measurements on a sufficiently large scale are difficult and expensive. Proxies for CH4 emissions can provide an alternative, but each approach has limitations. Negussie et al. (2019)<ref>Negussie, E., González Recio, O., de Haas, Y., Gengler N., Soyeurt, H., Peiren, N., Pszczola, M., Garnsworthy, P., Battagin, M., Bayat, A., Lassen, J., Yan, T., Boland, T., Kuhla, B., Strabel, T., Schwarm, A., Vanlierde, A., and Biscarini, F. 2019. Machine learning ensemble algorithms in predictive analytics of dairy cattle methane emission using imputed versus non-imputed datasets. 7th GGAA – Greenhouse Gas and Animal Agriculture Conference held from August 4th to 8th, Iguassu Falls/Brazil. Oral communication, Book of Abstracts Page 40. <nowiki>http://www.ggaa2019.org/sites/default/files/proceedings-ggaa2019.pdf</nowiki></ref> recently showed the potential of proxies proxies that are easy to record in the farm. These proxies can be gathered in most farms and are a realistic threshold accuracy that can be obtained without more fancy proxies. Several techniques have been developed for the measurement of CH4 emissions from ruminants, with varying degrees of accuracy (see reviews by Cassandro et al., 2013<ref>Cassandro, M., Mele, M., Stefanon, B.. 2013. Genetic aspects of enteric methane emission in livestock ruminants. Italian J. Anim. Sci. 12:e73: 450-458.</ref> and Hammond et al., 2016A<ref>Hammond, K.J., Crompton, L.A., Bannink, A., Dijkstra, J., Yáñez-Ruiz, D.R., O’Kiely, P., Kebreab, E., Eugenè, M.A., Yu, Z., Shingfield, K.J., Schwarm, A., Hristov, A.N., and Reynolds, C.K. 2016A. Review of current in vivo measurement techniques for quantifying enteric methane emission from ruminants. Anim. Feed Sci. Technol. 219:13–30. doi:10.1016/j.anifeedsci.2016.05.018.</ref>), but routine individual measurements on a large scale (a requisite for genetic selection) have proven to be difficult and expensive (Pickering et al., 2015<ref>Pickering, N.K., Oddy, V.H., Basarab, J.A., Cammack, K., Hayes, B J., Hegarty, R.S., McEwan, J.C., Miller, S., Pinares, C., and de Haas, Y. 2015. Invited review: Genetic possibilities to reduce enteric methane emissions from ruminants. Animal 9:1431-1440.</ref>; Negussie et al., 2016<ref>Negussie E., Lehtinen, J., Mäntysaari, P., Liinamo, A-E., Mäntysaari, E., and Lidauer, M.. 2016. Non-invasive individual methane measurements in dairy cows using photoacoustic infrared spectroscopy technique. 6th Greenhouse Gases Animal Agriculture Conference (GGAA2016) 14-18 February 2016. Melbourne, Australia. Abstract. p62.</ref>). Therefore, identifying proxies (i.e. indicators or indirect traits) that are correlated to CH4 emission, but which are easy and relatively low-cost to record on a large scale, is a much needed alternative. Proxies might be less accurate, but could be measured repeatedly to reduce random noise. The (potential) proxies range from simple and low-cost measurements such as body weight, to high-throughput milk MIR, to more demanding measures like rumen morphology, rumen metabolites or microbiome profiling.

Combining proxies that are easy to measure and cheap to record could provide predictions of CH4 emissions that are sufficiently accurate for selection and management of cows with low CH4 emissions.

== Available Proxies ==
A large array of CH4 proxies differing widely in accuracy and applicability under different conditions have been reported. The ideal proxy would be highly phenotypically and genetically correlated with CH4 emissions and could easily, and potentially repeatedly, be measured on a large scale. A systematic summary and assessment of existing knowledge is needed for the identification of robust and accurate CH4 proxies for future use. Table 5 summarizes proxies for CH4 production, and Table 6 summarizes results from combining proxies to improve predictability of proxies for CH4 prediction.
{| class="wikitable"
|+
!Proxy
!Description / conclusion
!Reference
|-
| colspan="3" |'''(1) Feed intake and feeding behavior'''
|-
|Dry Matter Intake (DMI)
|DMI predict MeP with R2= 0.06-0.64, and ME intake predict MeP with R2= 0.53-0,55
| Ellis et al. (2007);<ref>Ellis, J.L., Kebreab, E., Odongo, N.E., McBride, B.W., Okine, E.K., and France, J. 2007. Prediction of methane production from dairy and beef cattle. J. Dairy Sci. 90:3456–3466.</ref>
Mills et al. (2003)<ref>Mills, J.A.N., Kebreab, E., Yates, C.M., Crompton, L.A., Cammell, S.B., Dhanoa, M.S., Agnew, R.E., and France, J. 2003. Alternative approaches to predicting methane emissions from dairy cows. J. Anim. Sci. 81:3141–3150.</ref>;

Negussie et al. (2019)<ref>Negussie, E., González Recio, O., de Haas, Y., Gengler N., Soyeurt, H., Peiren, N., Pszczola, M., Garnsworthy, P., Battagin, M., Bayat, A., Lassen, J., Yan, T., Boland, T., Kuhla, B., Strabel, T., Schwarm, A., Vanlierde, A., and Biscarini, F. 2019. Machine learning ensemble algorithms in predictive analytics of dairy cattle methane emission using imputed versus non-imputed datasets. 7th GGAA – Greenhouse Gas and Animal Agriculture Conference held from August 4th to 8th, Iguassu Falls/Brazil. Oral communication, Book of Abstracts Page 40. <nowiki>http://www.ggaa2019.org/sites/default/files/proceedings-ggaa2019.pdf</nowiki></ref>
|-
|Gross Energy intake (GE)
|Predict MeP with RMSPE= 3.01.
| Moraes et al. (2014)<ref name=":0">Moraes, L.E., Strathe, A.B., Fadel, J.G., Casper, D.P., and Kebreab, E. 2014. Prediction of enteric methane emissions from cattle. Glob. Change Biol. 20:2140–2148.</ref>
|-
|Feeding behavior
| Magnitude and direction of relation to MeP varies across studies
|Nkrumah et al. (2006);<ref>Nkrumah, J.D., Okine, E.K., Mathison, G.W., Schmid, K., Li, C., Basarab, J.A., Price, M.A., Wang, Z., and Moore, S.S. 2006. Relationships of feedlot feed efficiency, performance, and feeding behavior with metabolic rate, methane production, and energy partitioning in beef cattle. J. Anim. Sci. 84:145-153.</ref>
Jonker et al., 2014<ref>Jonker, A., Molano, G., Antwi, C., Waghorn, G.. 2014. Feeding lucerne silage to beef cattle at three allowances and four feeding frequencies affects circadian patterns of methane emissions, but not emissions per unit of intake. Anim. Prod. Sci.54:1350-1353.</ref>
|-
| Rumination time
|High rumination relates to more milk, consume more concentrate and produce more CH4, lower RMP and MeI
| Watt et al. (2015)<ref>Watt, L.J., Clark, C.E.F., Krebs, G.L., Petzel, C.E., Nielsen, S., and Utsumi, S.A. 2015. Differential rumination, intake, and enteric methane production of dairy cows in a pasture-based automatic milking system. J. Dairy Sci. 98:7248–7263.</ref>;
López- Paredes et al. (2020)<ref>Lopez-Paredes, J., Goiri, I., Atxaerandio, R., García-Rodríguez, A., Ugarte, E., Jiménez-Montero, J.A., Alenda, R and González-Recio, O. 2020. Mitigation of greenhouse gases in dairy cattle via genetic selection (i): Genetic parameters of direct methane using non-invasive methods and its proxies. J. Dairy Sci. 103.</ref>
|-
|Rumen microbiome
|The metagenome can predict DMI, and classify high vs low intakes
|Delgado et al. (2019)<ref>Delgado, B., Bach A., Guasch I., González C, Elcoso G., Pryce J.E., Gonzalez-Recio O. (2019).Whole rumen metagenome sequencing allows classifying and predicting feed efficiency and intake levels in cattle. Scientific Reports 9: 11. doi:10.1038/s41598-018-36673-w</ref>
|-
| colspan="3" |'''(2) Rumen function, metabolites and microbiome'''
|-
|Dietary antimethanogenic compounds
|Inhibitors of the enzyme methyl coenzyme-M reductase: bromochoromethane; chloroform; 3- nitrooxypropanol (not always)
|Denman et al., 2007;<ref>Denman, S.E., Tomkins, N.W., and McSweeney, C.S. 2007. Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane. FEMS Microbiol. Ecol. 62:313-322.</ref>
Knight et al., 2011;<ref>Knight, T., Ronimus, R.S., Dey, D., Tootill, C., Naylor, G., Evans, P., Molano, G., Smith, A., Tavendale, M., Pinares-Patiño, C.S., and Clark, H. 2011. Chloroform decreases rumen methanogenesis and methanogen populations without altering rumen function in cattle. Anim. Feed Sci. Technol. 166-167:101-112.</ref>

Haisan et al., 2014<ref>Haisan, J., Sun, Y., Guan, L.L., Beauchemin, K.A., Iwaasa, A., Duval, S., Barreda, D.R., and Oba, M. 2014. The effects of feeding 3-nitrooxypropanol on methane emissions and productivity of Holstein cows in mid lactation. J. Dairy Sci. 97:3110-3119.</ref>;

Romero-Perez et al., 2014<ref>Romero-Perez, A., Okine, E.K., McGinn, S.M., Guan, L.L., Oba, M., Duval, S.M., Kinderman,n M., and Beauchemin, K.A. 2014. The potential of 3-nitrooxypropanol to lower enteric methane emissions from beef cattle. J. Anim. Sci. 92:4682-4693.</ref>
|-
|Dietary antimicrobial compounds
|Induce reductions in both MeP and methanogens numbers: nitrates, anacardic acid (cashew nut shell liquid), monensin, isobutyrate
|Iwamoto et al., 2002;<ref>Iwamoto, M., Asanuma, N., and Hin,o T. 2002. Ability of Selenomonas ruminantium, Veillonella parvula, and Wolinella succinogenes to reduce nitrate and nitrite with special reference to the suppression of ruminal methanogenesis. Anaerobe. 8:209-215.</ref>
Kubo et al., 1993<ref>Kubo, I., Muroi, H., Himejima, M., Yamagiwa, Y., Mera, H., Tokushima, K., Ohta, S., and Kamikawa, T. 1993. Structure-antibacterial activity relationships of anacardic acids. J. Agric. Food Chem. 41:1016-1019.</ref>;

van Zijderveld et al., 2010<ref>Van Zijderveld, S.M., Gerrits, W.J.J., Apajalahti, J.A., Newbold, J.R., Dijkstra, J., Leng, R A., and Perdok, H.B. 2010. Nitrate and sulfate: effective alternative hydrogen sinks for mitigation of ruminal methane production in sheep. J. Dairy Sci. 93:5856-5866.</ref>;

Veneman et al., 2015<ref>Veneman, J.B., Muetzel, S., Hart, K.J., Faulkner, C.L., Moorby, J.M., Perdok, H.B., and Newbold, C.J. 2015. Does Dietary Mitigation of Enteric Methane Production Affect Rumen Function and Animal Productivity in Dairy Cows? PLoS ONE 10(10): e0140282. doi: 10.1371/journal.pone.0140282</ref>;

Shinkai et al., 2012<ref>Shinkai, T., Enishi, O., Mitsumori, M., Higuchi, K., Kobayashi, Y., Takenaka, A., Nagashima, K., and Mochizuki, M. 2012. Mitigation of methane production from cattle by feeding cashew nut shell liquid. J. Dairy Sci. 95:5308-5316.</ref>;

Wang et al., 2015<ref>Wang, C., Liu, Q., Zhang, Y.L., Pei, C.X., Zhang, S.L., Wang, Y.X., Yang, W.Z., Bai, Y.S., Shi, Z.G., and Liu, X.N. 2015. Effects of isobutyrate supplementation on ruminal microflora, rumen enzyme activities and methane emissions in Simmental steers. J. Anim. Physiol. Anim. Nutr. (Berl). 99:123-131.</ref>
|-
|Rumen microbiome profile
|High Fibrobacteres, Quinella ovalis and Veillonellaceae and low Ruminococcaceae, Lachnospiraceae and Clostridiales associate with low- CH4 phenotypes and high propionate
Protozoa concentration
|Kittelmann et al., 2014<ref>Kittelmann, S., Pinares-Patiño, C.S., Seedorf, H., Kirk, M.R., Ganesh, S., McEwan, J.C., and Janssen, P.H. 2014. Two different bacterial community types are linked with the low-methane emission trait in sheep. PLoS One 9:e103171.</ref>;
Wallace et al., 2014<ref>Wallace, R. ., Rooke, J A., Duthie, C.-A., Hyslop, J.J., Ross, D.W., McKain, N., de Souza, S.M., Snelling, T.J., Waterhouse, A., and Roehe, R. 2014. Archaeal abundance in post-mortem ruminal digesta may help predict methane emissions from beef cattle. Sci. Rep. 4:5892.</ref>;

Sun et al., 2015<ref>Sun, X., Henderson, G., Cox, F., Molan,o G., Harrison, S.J., Luo, D., Janssen, P.H., and Pacheco, D. 2015. Lambs Fed Fresh Winter Forage Rape (Brassica napus L.) Emit Less Methane than Those Fed Perennial Ryegrass (Lolium perenne L.), and Possible Mechanisms behind the Difference. PLoS One 10(3):e0119697: DOI: 10.1371/journal.pone.0119697</ref>;

Guyader et al., 2014<ref>Guyader, J., Eugène, M., Nozière, P., Morgavi, D.P., Doreau, M., and Martin, C. 2014. Influence of rumen protozoa on methane emission in ruminants: a meta-analysis approach. Animal 8:1816-1825.</ref>
|-
|Rumen microbiome profile
|Predict MeP with R2 up to 0.55
|Ross et al. 2013a<ref>Ross, E. M., P.J . Moate, L.C. Marett, B.G. Cocks and B.J. Hayes. 2013a. Investigating the effect of two methane-mitigating diets on the rumen microbiome using massively parallel sequencing. J. Dairy Sci. 96:6030–6046.</ref>;
Ross et al. (2013b)<ref>Ross, E. M., Moate, P. J., Marett, L.C., Cocks, B.G., and Hayes, B.J. 2013b. Metagenomic Predictions: From Microbiome to Complex Health and Environmental Phenotypes in Humans and Cattle. PLoS One DOI: 10.1371/journal.pone.0073056.</ref>
|-
|Microbial genes
|20 (out of 3970 identified) related to CH4 emissions
|Roehe et al. (2016)<ref>Roehe R., Dewhurst, R.J., Duthie, C-A., Rooke, J.A., McKain, N., Ross, D.W., Hyslop, J.J., Waterhouse, A., Freeman, T.C., Watson, M., and Wallace, R.J. 2016. Bovine Host Genetic Variation Influences Rumen Microbial Methane Production with Best Selection Criterion for Low Methane Emitting and Efficiently Feed Converting Hosts Based on Metagenomic Gene Abundance. PLoS Genet 12(2): e1005846. doi:10.1371/journal.pgen.1005846.</ref>
|-
|Rumen volume (Xray Computed Tomography) and retention time
|Low-MeY sheep had smaller rumens. Faster passage= less time to ferment substrate - explained 28% of variation in MeP
|Pinares Patiño et al., 2003<ref>Pinares-Patiño C.S., Hickey, S.M., Young, E.A., Dodds, K.G., MacLean, S., Molano, G., Sandoval, E., Kjestrup, H., Harland, R., Pickering, N.K., and McEwan, J.C. 2013. Heritability estimates of methane emissions from sheep. Animal 7: 316–321.</ref>;
Goopy et al., 2014<ref>Goopy, J.P., Donaldson, A., Hegarty, R., Vercoe, P.E., Haynes, F., Barnett, M., and Oddy, V.H. 2014. Low-methane yield sheep have smaller rumens and shorter rumen retention time. Br. J. Nutr. 111:578-585.</ref>;

Okine et al. (1989)<ref>Okine, E. ., Mathiso,n G.W., and Hardin, R.T. 1989. Effects of changes in frequency of reticular contractions on fluid and particulate passage rates in cattle. J. Anim. Sci. 67:3388–3396.</ref>
|-
|Blood triiodothyronine concentration
|Reduced MeY
|Barnett et al. (2012)<ref>Barnett, M.C., Goopy, J.P., McFarlane, J.R., Godwin, I.R., Nolan, J.V., and Hegarty, R.S. (2012). Triiodothyronine influences digesta kinetics and methane yield in sheep. Anim. Prod. Sci. 52:572-577.</ref>
|-
|Acetate to propionate ratio in ruminal fluid
|Positively associated with CH4 emissions, but not confirmed in all studies, sometimes opposite relation
|Mohammed et al., 2011;<ref name=":1">Mohammed, R., McGinn, S.M., and Beauchemin, K.A. 2011. Prediction of enteric methane output from milk fatty acid concentrations and rumen fermentation parameters in dairy cows fed sunflower, flax, or canola seeds. J. Dairy Sci. 94:6057–6068.</ref>
Fievez et al., 2012<ref>Fievez V., Colma,n E., Castro-Montoya, J.M., Stefanov, I., and Vlaeminck, B. 2012. Milk odd- and branched-chain fatty acids as biomarkers of rumen function – An update. Anim. Feed. Sci. Technol. 172:51–65.</ref>;

Chung et al., 2011<ref>Chung, Y.-H., Walker, N.D., McGinn, S.M., and Beauchemin, K.A. 2011. Differing effects of 2 active dried yeast (Saccharomyces cerevisiae) strains on ruminal acidosis and methane production in nonlactating dairy cows. J. Dairy Sci. 94:2431–2439.</ref>;

Van Zijderveld et al., 2010<ref>Van Zijderveld, S.M., Gerrits, W.J.J., Apajalahti, J.A., Newbold, J.R., Dijkstra, J., Leng, R A., and Perdok, H.B. 2010. Nitrate and sulfate: effective alternative hydrogen sinks for mitigation of ruminal methane production in sheep. J. Dairy Sci. 93:5856-5866.</ref>
|-
| colspan="3" |'''(3) Milk production and composition'''
|-
|Modelling approach
|Doubling milk production only adds 5 kg to the MeP and so greatly reduces MeY
|Kirchgessner et al. (1995)<ref>Kirchgessner M., Windisch, W., and Muller, H.L. 1995. Nutritional factors for the quantification of methane production. In: Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction. Proceedings 8th International Symposium on Ruminant Physiology (eds W. Von Engelhardt, S. Leonhard-Marek, G. Breves and D. Giesecke). Reproduction Proceedings 8th International Symposium on Ruminant Physiology. Ferdinand Enke Verlag, Stuttgart, Germany. pp. 333-348.</ref>;
Hristov et al. (2014)<ref>Hristov, A.N., Johnson, K.A., and Kebreab, E, 2014. Livestock methane emissions in the United States. Proc. Natl. Aacad. Sci. 111:E1320; doi:10.1073/pnas.1401046111</ref>
|-
|Milk fat content
|key explanatory variable for predicting CH4: A moderate negative genetic correlation with infrared predicted
MeI: correlations MeP = 0,08 and MeI = - 0.13
|Moraes et al. (2014)<ref>Moraes, L.E., Strathe, A.B., Fadel, J.G., Casper, D.P., and Kebreab, E. 2014. Prediction of enteric methane emissions from cattle. Glob. Change Biol. 20:2140–2148.</ref>;
Kandel et al., 2014A<ref name=":2">Kandel, P.B., Vanderick, S., Vanrobays, M.L., Vanlierde, A., Dehareng, F., Froidmont, E., Soyeurt, H., and Gengler, N. 2014A. Consequences of selection for environmental impact traits in dairy cows. Page 19. (<nowiki>http://orbi.ulg.ac.be/bitstream/2268/164402/164401/NSABS162014_poster_Purna_abstract.pdf</nowiki>) I:n Proc. 19th National symposium on applied biological sciences, Gembloux, Belgium.</ref>, B<ref name=":3">Kandel, P.B., Vanderick, S., Vanrobays, M.L., Vanlierde, A., Dehareng, F., Froidmont, E., Soyeur,t H., and Gengler, N. 2014B. Consequences of selection for environmental impact traits in dairy cows. In: 10th World Congress on Genetics Applied to Livestock Production (WCGALP), 17-22 August, 2014. Vancouver, Canada.</ref>;

Vanlierde et al. (2015)<ref name=":4">Vanlierde, A., Vanrobays, M.L., Dehareng, F., Froidmont, E., Soyeurt, H., McParland, S., Lewis, E., Deighton, M.H., Grandl, F., Kreuzer, M., Grendler, B., Dardenne, P., and Gengler, N. 2015. Hot topic: Innovative lactation-stage-dependent prediction of methane emissions from milk mid-infrared spectra. J. Dairy Sci. 98:5740–5747.</ref>
|-
|Milk fat content
|A positive relationship between VFA proportions and methanogenesis is expected as a consequence of the common biochemical pathways; Dietary unsaturated fatty acids are negatively associated with CH4 emissions
|Vlaeminck et al., 2006<ref>Vlaeminck, B., Fievez, V., Cabrita, A.R.J., Fonseca, A.J.M., and Dewhurst, R.J. 2006. Factors affecting odd- and branched-chain fatty acids in milk: A review. Anim. Feed Sci. Technol. 131:389–417.</ref>;
Van Lingen et al., 2014<ref name=":5">Van Lingen H.J., Crompton, L.A., Hendriks, W.H., Reynolds, C.K., Dijkstra, J. 2014. Meta-analysis of relationships between enteric methane yield and milk fatty acid profile in dairy cattle. J. Dairy Sci. 97:7115-7132.</ref>
|-
|Milk protein yield
|Correlation with Mel = - 0.47 or -0.09, MeP = 0.53
|Kandel et al. (2014)<ref name=":2" />;
Vanlierde et al. (2015)<ref name=":4" />
|-
|Lactose
|Variable correlations: MeP = 0,33; MeI = - 0.21; R = 0.19 for CH4 emission
|Miettinen and Huhtanen (1996)<ref>Miettinen, H., and Huhtanen, P. 1996. Effects of the ration of ruminal propionate to butyrate on milk yield and blood metabolites in dairy cows. J. Dairy Sci. 79:851–861.</ref>;
Dehareng et al. (2012)<ref>Dehareng, F., Delfosse, C., Froidmont, E., Soyeurt, H., Martin, C., Gengler, N., Vanlierde, A., and Dardenne, P. 2012. Potential use of milk mid-infrared spectra to predict individual methane emission of dairy cows. Animal 6:1694-701.</ref>
|-
|Somatic cell score
|Genetic correlation with infrared predicted MeI: R = 0.07
|Kandel et al. (2014A<ref name=":2" />, B<ref name=":3" />)
|-
|Prediction equations Milk FA and CH4 emissions, including from MIR data
|R² ranged between 47 and 95%; relationships between the individual milk FA and MeP differed considerably and the correlations between CH4 and milk FA vary throughout the lactation
|Chilliard et al. (2009)<ref>Chilliard Y., Martin ,C., Rouel, J., and Doreau, M. 2009. Milk fatty acids in dairy cows fed whole crude linseed, extruded linseed, or linseed oil, and their relationship with methane output. J. Dairy Sci. 92:5199-5211.</ref>;
Delfosse et al. (2010)<ref>Delfosse, O., Froidmont, E., Fernandez Pierna, J. A., Martin, C., and Dehareng, F. 2010. Estimation of methane emissions by dairy cows on the basis of milk composition. In: Greenhouse Gases and Animal Agriculture Conference. 2010; GGAA2010: 4. Greenhouse Gases and Animal Agriculture Conference, Banff, CAN, 2010-10-03-2010-10-08, 60-61.</ref>;

Castro-Montoya et al. (2011)<ref>Castro Montoya, J., Bhagwat, A.M., Peiren, N., De Campeneere, S., De Baets, B., and Fievez, V. 2011. Relationships between odd- and branched-chain fatty acid profiles in milk and calculated enteric methane proportion for lactating dairy cattle. Anim. Feed Sci. Technol.166:596–602.</ref>;

Dijkstra et al. (2011)<ref>Dijkstra, J., van Zijderveld, S.M., Apajalahti, J.A., Bannink, A., Gerrits, W.J.J., Newbold, J.R., Perdok, H.B.,, and Berends, H. 2011. Relationships between methane production and milk fatty acid profiles in dairy cattle. Anim. Feed Sci. Technol. 166–167:590–595.</ref>;

Kandel et al. (2013)<ref>Kandel, P.B., Vanrobays, M.L., Vanlierde, A., Dehareng, F., Froidmont, E., Dardenne, P., Lewis, E., Buckley, F., Deighton, M.H., McParland, S. and Gengler, N., 2013. Genetic parameters for methane emissions predicted from milk mid-infrared spectra in dairy cows. J. Dairy Sci. 95(E-1):p.388.</ref>;

Mohammed et al. (2011)<ref name=":1" />;

Van Lingen et al. (2014)<ref name=":5" />;

Williams et al. (2014)<ref>Williams, S.R.O., Williams, B., Moate, P.J., Deighton, M.H., Hannah, M.C., and Wales, W.J. 2014. Methane emissions of dairy cows cannot be predicted by the concentrations of C8:0 and total C18 fatty acids in milk. Anim. Prod. Sci. 54:1757–1761. <nowiki>http://dx.doi.org/10.1071/AN14292</nowiki>.</ref>;

Dijkstra et al. (2016)<ref>Dijkstra, J., van Zijderveld, S.M., Apajalahti, J.A., Bannink, A., Gerrits, W.J.J., Newbold, J.R., Perdok, H.B.,, and Berends, H. 2011. Relationships between methane production and milk fatty acid profiles in dairy cattle. Anim. Feed Sci. Technol. 166–167:590–595.</ref>;

Rico et al. (2016)<ref>Rico D.E., Chouinard, P.Y., Hassanat, F., Benchaar, C., and Gervais, R. 2016. Prediction of enteric methane emissions from Holstein dairy cows fed various forage sources. Animal 10:203-211. doi:10.1017/S1751731115001949</ref>;

Van Gastelen and Dijkstra (2016)<ref>Van Gastelen, S., and Dijkstra, J.. 2016. Prediction of methane emission from lactating dairy cows using milk fatty acids and mid-infrared spectroscopy. J. Sci. Food Agric. 96:3963-3968. DOI: 10.1002/jsfa.7718.</ref>;

Vanrobays et al. (2016);<ref>Vanrobays, M.-L., Bastin, C., Vandenplas, J., Hammami, H., Soyeurt, H., Vanlierde, A., Dehareng, F., Froidmont, E., and Gengler, N. 2016. Changes throughout lactation in phenotypic and genetic correlations between methane emissions and milk fatty acid contents predicted from milk mid-infrared spectra. J. Dairy Sci. 99:1–14. <nowiki>http://dx.doi.org/10.3168/jds.2015-10646</nowiki></ref>

Bougoin et al., (2019)<ref>Bougouin, A., Appuhamy, J.A.D.R.N., Ferlay, A., Kebreab, E., Martin, C., Moate, P.J., Benchaar, C., Lund, P., and Eugène, M. 2019. Individual milk fatty acids are potential predictors of enteric methane emissions from dairy cows fed a wide range of diets: Approach by meta-analysis. J. Dairy Sci. 102:10616–10631. DOI: <nowiki>http://doi.org/10.3168/jds.2018-15940</nowiki></ref>
|-
| colspan="3" |'''(4) Hind-gut and feces'''
|-
|Whole tract digestibility (potential as supporting factors in the prediction of enteric CH4 emissions)
|Main effects relate to rumen (see above), but energy digestibility as a supporting factor to GE intake improved the accuracy of CH4 prediction, despite the fact that there was no direct linear relationship between energy digestibility and MeY and in % of GE intake
|Yan et al., 2009 C<ref name=":6">Yan, T., Porter, M.G., and Mayne, S.C. 2009. Prediction of methane emission from beef cattle using data measured in indirect open-circuit respiration calorimeters. Animal 3:1455-1462.</ref>
|-
|Ratio of acetic and butyric acid divided by propionic acid
|Methane yield positive relation
|Moss et al., 2000<ref>Moss A.R., Jouany, J.P., and Newbold, J. 2000. Methane production by ruminants: Its contribution to global warming. Annal. Zootech. 49:231-253.</ref>
|-
| colspan="3" |'''(5) Whole animal measurements'''
|-
|Body weight and conformation
|Prediction models; primary predictor for enteric MeP
|Moraes et al. (2014)<ref name=":0" />;
Holter and Young, 1992; <ref>Holter J.B., and Young, A.J. 1992. Methane production in dry and lactating Holstein cows. J. Dairy Sci. 75:2165–2175.</ref>

Yan et al., 2009<ref name=":6" />
|-
|Body weight
|Relationship with MeI: r = 0.44; relationship between body weight and rumen capacity
|Antunes-Fernandes et al. (2016)<ref>Antunes-Fernandes, E.C., van Gastelen, S., Dijkstra, J., Hettinga K.A., and Vervoort, J. 2016. Milk metabolome relates enteric methane emission to milk synthesis, and energy metabolism pathways. J. Dairy. Sci. 99:6251-6262.</ref>;
Demment and Van Soest, 1985<ref>Demment, M.W., and Van Soest, P.J. 1985. A nutritional explanation for body-size patterns of ruminant and nonruminant herbivores. Am. Nat. 125:641–672.</ref>
|-
|Body weight
|Key explanatory variable for enteric MeP
|No reference available
|-
|Conformation traits: affects enteric MeP
|Indicators for rumen volume (via feed intake and rumen passage rates); BCS
|Agnew and Yan, 2000<ref>Agnew, R.E. and Yan, T. 2000. The impact of recent research on energy feeding systems for dairy cattle. Livest. Prod. Sci. 66:197-215.</ref>
|-
|Lactation stage
|Complementary proxy
|Vanlierde et al. (2015)<ref name=":4" />
|}Table 5. Available methane proxies include: (1) feed intake and feeding behaviour, (2) rumen function, metabolites and microbiome, (3) milk production and composition, (4) hind-gut and faeces, and (5) measurements at the level of the whole animal. It is evident that no single proxy offers a good solution in terms of all of these attributes, though the low cost and high throughput make milk MIR a good candidate for further work on refining methods, improving calibrations and exploring combinations with other proxies.

Section 20: Proxies Discussion

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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]].

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The greatest limitation of proxies today is the lack of robustness in their general applicability. Future efforts should therefore be directed towards developing combinations of proxies that are robust and applicable across diverse production systems and environments. Here we present the present status of the knowledge of proxies and their predictive value for CH4 emission. Proxies related to body weight or milk yield and composition are relatively simple, low-cost, high throughput, and are easy to implement in practice. In particular, DMI and milk MIR, along with covariates such as lactation stage, are a promising option for prediction of CH4 emission in dairy cows. No single proxy was found to accurately predict CH4, whilst combinations of two or more proxies are likely to be a better solution. Combining proxies can increase the accuracy of predictions by up to 15 - 35%, mainly because different proxies describe independent sources of variation in CH4 and one proxy can correct for shortcomings in the other(s). One plausible strategy could be to increase animal productive efficiency whilst reducing CH4 emissions per animal. This could be achieved by reducing MeY and/or decreasing DMI provided that there is no concomitant reduction in productivity or increase in feed consumption (Pickering et al., 2015<ref>Pickering, N.K., Oddy, V.H., Basarab, J.A., Cammack, K., Hayes, B J., Hegarty, R.S., McEwan, J.C., Miller, S., Pinares, C., and de Haas, Y. 2015. Invited review: Genetic possibilities to reduce enteric methane emissions from ruminants. Animal 9:1431-1440.</ref>).

=== Combining diet-based measurements with other proxies for methane emissions ===
Feed intake appears a reasonably adequate predictor of MeP: generally, heavier animals have higher maintenance requirements, so eat more and produce more CH4. However, a substantial level of variation is left unaccounted for. This suggests that more detailed information on dietary composition is needed. This is also important when one wants to account for MeP on diets of similar DMI but of different nutrient profiles.

The prediction accuracy of MeP strongly depends on the accuracy of quantifying the VFA produced in the rumen (Alemu et al., 2011<ref>Alemu, A.W., Dijkstra, J., Bannink, A., France, J., and Kebreab, E. 2011. Rumen stoichiometric models and their contribution and challenges in predicting enteric methane production. Anim. Feed Sci. Technol. 166-167:761-778.</ref>). The type of VFA formed during rumen fermentation depends on the type of substrate fermented (Bannink et al., 2011<ref>Bannink, A., van Schijndel, M.W., and Dijkstra, J. 2011. A model of enteric fermentation in dairy cows to estimate methane emission for the Dutch National Inventory Report using the IPCC Tier 3 approach. Anim. Feed Sci. Technol. 166-167:603-618.</ref>), such as the dietary content of neutral detergent fiber and starch. The type of substrate fermented thus appears a useful factor for predicting MeP (Ellis et al., 2007<ref>Ellis, J.L., Kebreab, E., Odongo, N.E., McBride, B.W., Okine, E.K., and France, J. 2007. Prediction of methane production from dairy and beef cattle. J. Dairy Sci. 90:3456–3466.</ref>), indicating that including a description of variation in dietary quality caused by nutritional factors results in improved prediction accuracy of CH4 emission (Ellis et al., 2010<ref>Ellis, J.L., Bannink, A., France, J., Kebreab, E., and Dijkstra, J. 2010. Evaluation of enteric methane prediction equations for dairy cows used in whole farm models. Glob. Change Biol. 16:3246–3256.</ref>; Moraes et al., 2014<ref name=":0">Moraes, L.E., Strathe, A.B., Fadel, J.G., Casper, D.P., and Kebreab, E. 2014. Prediction of enteric methane emissions from cattle. Glob. Change Biol. 20:2140–2148.</ref>).

=== Rumen ===
When feed intake is kept constant, a higher rumen capacity results in a lower passage rate (Demment and Van Soest, 1985<ref>Demment, M.W., and Van Soest, P.J. 1985. A nutritional explanation for body-size patterns of ruminant and nonruminant herbivores. Am. Nat. 125:641–672.</ref>), resulting in a higher MeP (Moraes et al., 2014<ref name=":0" />). Proxies based on rumen samples are generally poor to moderately accurate predictors of CH4, and are costly and difficult to measure routinely on-farm. VFA are a proxy for rumen CH4 emissions. Using rumen fermentation data obtained from in vitro gas production, Moss et al. (2000)<ref>Moss A.R., Jouany, J.P., and Newbold, J. 2000. Methane production by ruminants: Its contribution to global warming. Annal. Zootech. 49:231-253.</ref> reported a negative linear relationship between CH4 production and the ratio of (acetic + butyric acid)/propionic acid. However, by combining different information sources, either related to feed intake or to the impact of feed intake on the VFA composition, a better proxy with an improved accuracy can be achieved. This way, the prediction equation for CH4 production can be optimized (higher accuracy).

The relationship between rumen methanogen abundance and methanogenesis is less clear when changes in enteric CH4 emissions are modulated by diet or are a consequence of selecting phenotypes related to feed efficiency or MeY. Whereas in some reports there was a significant positive relationship (Aguinaga Casanas et al., 2015<ref name=":1">Aguinaga Casanas, M.A., , N., Krattenmacher, N., Thalle,r G., Metges, C.C., and Kuhla, B. 2015. Methyl-coenzyme M reductase A as an indicator to estimate methane production from dairy cows. J. Dairy Sci. 98:4074-4083.</ref>; Arndt et al., 2015<ref>Arndt, C., Powell, J.M., Aguerre, M.J., Crump, P.M., and Wattiaux, M.A. 2015. Feed conversion efficiency in dairy cows: Repeatability, variation in digestion and metabolism of energy and nitrogen, and ruminal methanogens. J. Dairy Sci. 98:3938-3950.</ref>; Sun et al., 2015<ref>Sun, X., Henderson, G., Cox, F., Molan,o G., Harrison, S.J., Luo, D., Janssen, P.H., and Pacheco, D. 2015. Lambs Fed Fresh Winter Forage Rape (Brassica napus L.) Emit Less Methane than Those Fed Perennial Ryegrass (Lolium perenne L.), and Possible Mechanisms behind the Difference. PLoS One 10(3):e0119697: DOI: 10.1371/journal.pone.0119697</ref>; Wallace et al., 2015<ref name=":2">Wallace, R., Rooke, J., McKain, N., Duthie, C.-A., Hyslop, J., Ross, D., Waterhouse, A., Watson, M., and Roehe, R. 2015. The rumen microbial metagenome associated with high methane production in cattle. BMC Genomics. 16:839.</ref>), in many others the concentration of methanogens was unrelated to methanogenesis (Morgavi et al., 2012<ref>Morgavi, D.P., Martin, C., Jouany, J.P., and Ranilla, M.J. 2012. Rumen protozoa and methanogenesis: not a simple cause-effect relationship. Br. J. Nutr. 107:388-397. 10.1017/S0007114511002935.</ref>; Kittelmann et al., 2014<ref>Kittelmann, S., Pinares-Patiño, C.S., Seedorf, H., Kirk, M.R., Ganesh, S., McEwan, J.C., and Janssen, P.H. 2014. Two different bacterial community types are linked with the low-methane emission trait in sheep. PLoS One 9:e103171.</ref>; Shi et al., 2014<ref name=":3">Shi W., Moon, C.D., Leahy, S.C., Kang, D., Froula, J., Kittelmann, S., Fan, C., Deutsch, S., Gagic, D., Seedorf, H., Kelly, W.J., Atua, R., Sang, C., Soni, P., Li, D., Pinares-Patiño, C.S., McEwan, J.C., Janssen, P.H., Chen, F., Visel, A., Wang, Z., Attwood, G.T., and Rubin, E.M. 2014. Methane yield phenotypes linked to differential gene expression in the sheep rumen microbiome. Genome Res. Doi:10.1101/gr.168245.113.</ref>; Bouchard et al., 2015<ref name=":4">Bouchard, K., Wittenberg, K.M., Legesse, G., Krause, D.O., Khafipour, E., Buckley, K.E., and Ominski, K.H. 2015. Comparison of feed intake, body weight gain, enteric methane emission and relative abundance of rumen microbes in steers fed sainfoin and lucerne silages under western Canadian conditions. Grass a Forage Sci. 70:116-129.</ref>). Bouchard et al. (2015)<ref name=":4" /> even reported a reduction in methanogens withoutsignificant decrease in MeP for steers fed sainfoin silage. Sheep selected for high or low MeY showed no differences in methanogen abundance, though there was a strong correlation with expression of archaeal genes involved in methanogenesis (Shi et al., 2014<ref name=":3" />).

Hindgut and Feces: whole tract digestibility variables cannot serve as primary predictors for enteric MeP in cattle or sheep, but might be used as supporting factors to improve the accuracy of prediction of CH4 output.

=== Protozoa and other rumen microbes ===
Protozoa are net producers of H2 and their absence from the rumen is associated with an average reduction in enteric MeP of approximately 11% (Hegarty, 1999<ref>Hegarty, R.S. 1999. Reducing rumen methane emissions through elimination of rumen protozoa. Aust. J. Agric. Res. 50:1321-1327.</ref>; Morgavi et al., 2010<ref>Morgavi, D.P., Forano, E., Martin, C., and Newbold, C.J. 2010. Microbial ecosystem and methanogenesis in ruminants. Animal 4:1024-1036.</ref>; Newbold et al., 2015<ref>Newbold, C.J., de la Fuente, G., Belanche, A., Ramos-Morales, E., and McEwan, N. 2015. The role of ciliate protozoa in the rumen. Front. Microbiol. 6:1313.doi: 10.3389/fmicb.2015.01313</ref>). Using a database of 28 experiments and 91 dietary treatments, Guyader et al. (2014)<ref>Guyader, J., Eugène, M., Nozière, P., Morgavi, D.P., Doreau, M., and Martin, C. 2014. Influence of rumen protozoa on methane emission in ruminants: a meta-analysis approach. Animal 8:1816-1825.</ref> showed a significant decrease of 8.14 g CH4/kg DMI for each log unit reduction in rumen protozoal abundance. About 21% of experiments within this dataset reported CH4 changes unrelated to protozoal abundance, highlighting the multifactorial nature of methanogenesis. Roehe et al. (2016)<ref>Roehe R., Dewhurst, R.J., Duthie, C-A., Rooke, J.A., McKain, N., Ross, D.W., Hyslop, J.J., Waterhouse, A., Freeman, T.C., Watson, M., and Wallace, R.J. 2016. Bovine Host Genetic Variation Influences Rumen Microbial Methane Production with Best Selection Criterion for Low Methane Emitting and Efficiently Feed Converting Hosts Based on Metagenomic Gene Abundance. PLoS Genet 12(2): e1005846. doi:10.1371/journal.pgen.1005846.</ref> observed that the ranking of sire groups for CH4 emissions measured with respiration chambers was the same as that for ranking on archaea/bacteria ratio, providing further evidence that host control of archaeal abundance contributes to genetic variation in CH4 emissions - at least in some circumstances. Across a wide geographical range, the methanogenic archaea were shown to be highly conserved across the world (Henderson et al., 2015<ref>Henderson, G., Cox, F., Ganesh, S., Jonker, A., Young, W., Global Rumen Census Collaborators, and Janssen, P.H. 2015. Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range. Sci. Rep. 5:14567. doi:10.1038/srep14567</ref>). This universality and limited diversity could make it possible to mitigate CH4 emissions by developing strategies that target the few dominant methanogens. However, one clear limitation of metagenomic predictions compared to genomic predictions was that the microbiome of the host is variable - that is, it may change in response to diet or other environmental factors over time, whereas the hosts DNA remains constant.

=== Rumen microbial genes ===
These included genes involved in the first and last steps of methanogenesis: formylmethanofuran dehydrogenase subunit B (fmdB) and methyl-coenzyme M reductase alpha subunit (mcrA), which were 170 times more abundant in high CH4 emitting cattle. Whilst gene-centric metagenomics is not low-cost or high-throughput, these results point to potential future proxy approaches using low-cost gene chips.

The difference in gene expression activity as opposed to abundance was also reported by others (Popova et al., 2011<ref>Popova, M., Martin, C., Eugène, M., Mialon, M.M., Doreau, M., and Morgavi, D.P. 2011. Effect of fibre- and starch-rich finishing diets on methanogenic Archaea diversity and activity in the rumen of feedlot bulls. Anim. Feed Sci. Technol. 166-167:113-121.</ref>). However, there are also studies in which there was no relationship with gene expression (Aguinaga Casanas et al., 2015<ref name=":1" />). There are some methodological and experimental differences that might explain some of the apparent contradictions, such as the type of gene target and primers used for nucleic acid amplification. Effects are seen most clearly when the difference in MeP between groups of animals is large (e.g. Wallace et al. (2015) <ref name=":2" />used treatments that generated a 1.9-fold difference CH4 emissions).

=== Proxies based on measurements in milk ===
Milk yield alone does not provide a good prediction of MeP by dairy cows. Yan et al. (2010)<ref>Yan. T., Mayne, C.S., Gordon, F.G., Porter, M.G., Agnew, R.E., Patterson, D.C., Ferris, C.P., and Kilpatrick, D.J. 2010. Mitigation of enteric methane emissions through improving efficiency of energy utilization and productivity in lactating dairy cows. J. Dairy Sci. 93:2630–2638. doi: 10.3168/jds.2009-2929.</ref> indicated that CH4 as a proportion of GE intake or milk energy output was negatively related to milk production. It is less clear if MeY can be predicted from milk yield when making comparisons across studies.

Milk MIR spectroscopy is relatively inexpensive, rapid and already routinely used technology in milk recording systems to predict fat, protein, lactose and urea contents in dairy milk to assist farm management decisions and breeding. It can be used as a promising strategy to exploit the link between enteric CH4 emission from ruminants and microbial digestion in the rumen by assessing the signature of digestion in milk composition. Milk MIR data can be obtained through regular milk recording schemes, as well as, on a herd level, through analysis used for milk payment systems. Diverse milk phenotypes can be obtained by MIR spectrometry – including detailed milk composition (e.g. FA as reported by Soyeurt et al., 2011<ref>Soyeurt H., Dehareng, F., Gengler, N., McParland, S., Wall, E., Berry, D.P., Coffey, M., and Dardenne, P. 2011. Mid-infrared prediction of bovine milk fatty acids across multiple breeds, production systems and countries. J. Dairy Sci. 94:1657–1667.</ref>), technological properties of milk, and cow physiological status (De Marchi et al., 2014<ref>De Marchi, M., Toffanin, V., Cassandro, M., and Penasa, M. 2014. Invited review: Mid-infrared spectroscopy as phenotyping tool for milk traits. J. Dairy Sci. 97:1171–1186. <nowiki>http://dx.doi.org/10.3168/jds.2013-6799</nowiki>.</ref>; Gengler et al., 2016<ref>Gengler, N., Soyeurt, H., Dehareng, F., Bastin, C., Colinet, F., Hammami, H., Vanrobays, M.-L., Lainé, A., Vanderick, S., Grelet, C., Vanlierde, A., Froidmont, E., and Dardenne, P. 2016. Capitalizing on fine milk composition for breeding and management of dairy cows. J. Dairy Sci. 99:4071-4079.</ref>). Several of these novel traits (i.e. FA composition) have been identified as potential indicators of CH4 emission. Therefore, using MIR to predict MeP (Dehareng et al. 2012<ref name=":5">Dehareng, F., Delfosse, C., Froidmont, E., Soyeurt, H., Martin, C., Gengler, N., Vanlierde, A., and Dardenne, P. 2012. Potential use of milk mid-infrared spectra to predict individual methane emission of dairy cows. Animal 6:1694-701.</ref>; Vanlierde et al. 2013<ref>Vanlierde, A., Dehareng, F., Froidmont, E., Dardenne, P., Kandel, P.B., Gengler, N., Deighton, M.H., buckley, F., Lewis, E., McParland, S., Berry, D.P., and Soyeurt, H. 2013. Prediction of the individual enteric methane emission of dairy cows from milk-mid-infrared spectra. Advances in Animal Biosciences. 5th Greenhouse Gases Animal Agriculture Conference (GGAA2013) 23-26 June 2014. Dublin, Ireland. p 433.</ref>, 2015<ref name=":6">Vanlierde, A., Vanrobays, M.L., Dehareng, F., Froidmont, E., Soyeurt, H., McParland, S., Lewis, E., Deighton, M.H., Grandl, F., Kreuzer, M., Grendler, B., Dardenne, P., and Gengler, N. 2015. Hot topic: Innovative lactation-stage-dependent prediction of methane emissions from milk mid-infrared spectra. J. Dairy Sci. 98:5740–5747.</ref>; Van Gastelen and Dijkstra, 2016<ref name=":7">Van Gastelen, S., and Dijkstra, J.. 2016. Prediction of methane emission from lactating dairy cows using milk fatty acids and mid-infrared spectroscopy. J. Sci. Food Agric. 96:3963-3968. DOI: 10.1002/jsfa.7718.</ref>) is also a logical extension of its use to quantify the major milk components (i.e. fat, protein, casein, lactose, and urea) and minor components (e.g. FA). Dehareng et al. (2012)<ref name=":5" /> assessed the feasibility to predict individual MeP from dairy cows using milk MIR spectra. Their initial results suggest that this approach could be useful to predict MeP at the farm or regional scale, as well as to identify low-CH4 emitting cows. According to Van Gastelen and Dijkstra (2016)<ref name=":7" />, MIR spectroscopy has the disadvantage that it has a moderate predictive power for CH4 emission, both direct and indirect (i.e. via milk FA), and that it lacks the ability to predict important milk FA for CH4 prediction. They concluded that it may not be sufficient to predict MeP based on MIR alone. It is, however, possible to improve the accuracy of prediction through the combination of MIR with some animal characteristics such as lactation stage (Vanlierde et al., 2015<ref name=":6" />). The advantage of this latter development is that this type of prediction can be done on a very large scale inside a routine milk recording system (Vanlierde et al., 2015<ref name=":6" />).

=== Proxy: future developments and perspectives ===
There is currently limited consensus on which phenotype to use to lower the carbon footprint of milk production through genetic selection. This could be MeP, MeI or MeY. The direct goal would be CH4 production; the relationship with milk production and/or feed intake could be accounted for by including these in the final selection index or scheme. However, one might argue that it would be more effective/accurate to directly use milk production- or feed intakecorrected CH4 (e.g. CH4 intensity or yield) as breeding goal.

The analysis of proxies in terms of their attributes shows that proxies that are based on samples from the rumen or related to rumen sources are poor to moderately accurate predictors of CH4. In addition, these proxies are too costly and difficult for routine on-farm implementation. On the other hand, proxies related to BW, milk yield and composition (e.g. milk FA) are moderately to highly accurate predictors of CH4 and relatively simple, low-cost and easier to implement in practice (Cassandro et al.,2010<ref>Cassandro, M., Cecchinato, A., Battagin, M., Penasa, M., 2010. Genetic parameters of predicted methane production in Holstein Friesian cowsIn: Proc. 9th World Congr. on Genetics Applied to Livestock Production, Leipzig, Germany. . Page 181</ref>; Cassandro, 2013<ref>Cassandro, M. 2013. Comparing local and cosmopolitan cattle breeds on added values for milk and cheese production and their predicted methane emissions. Animal Genetic Resources/Ressources génétiques animales/Recursos genéticos animales, available on CJO2013. doi:10.1017/S2078 63361200077X. </ref>). Particularly, milk MIR and the prediction of CH4 based on milk MIR along with other covariates such as lactation stage is a promising alternative: that is accurate, cheaper and easy to be implemented in routine milk analysis at no extra cost.

Therefore, in the future advances in infrared, photoacoustic and related technologies will push the boundaries, particularly in focusing on developments of fast and portable technologies. Such developments will lead to better and promising proxies for CH4 that will enable a sizable throughput of CH4 phenotypes in dairy cows. Antunes-Fernandes et al. (2016)<ref>Antunes-Fernandes, E.C., van Gastelen, S., Dijkstra, J., Hettinga K.A., and Vervoort, J. 2016. Milk metabolome relates enteric methane emission to milk synthesis, and energy metabolism pathways. J. Dairy. Sci. 99:6251-6262.</ref> already presented the use of metabolomics on milk to better understand the biological pathways involved in CH4 production in dairy cattle. The techniques used in that study are not suitable for large scale measurements, but rapid developments in omics may offer tests and assay methodologies on blood, urine or milk samples that will provide an additional tool for developing new / additional proxies for CH4 emissions in dairy cattle.

Section 20: Merging and sharing data in genetic evaluations

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Section 20: Recommendations

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Section 20: Conclusions

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Section 20: Conclusions

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Section 20: Recommendations

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Section 20: Proxies Discussion

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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]].

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The greatest limitation of proxies today is the lack of robustness in their general applicability. Future efforts should therefore be directed towards developing combinations of proxies that are robust and applicable across diverse production systems and environments. Here we present the present status of the knowledge of proxies and their predictive value for CH4 emission. Proxies related to body weight or milk yield and composition are relatively simple, low-cost, high throughput, and are easy to implement in practice. In particular, DMI and milk MIR, along with covariates such as lactation stage, are a promising option for prediction of CH4 emission in dairy cows. No single proxy was found to accurately predict CH4, whilst combinations of two or more proxies are likely to be a better solution. Combining proxies can increase the accuracy of predictions by up to 15 - 35%, mainly because different proxies describe independent sources of variation in CH4 and one proxy can correct for shortcomings in the other(s). One plausible strategy could be to increase animal productive efficiency whilst reducing CH4 emissions per animal. This could be achieved by reducing MeY and/or decreasing DMI provided that there is no concomitant reduction in productivity or increase in feed consumption (Pickering et al., 2015<ref>Pickering, N.K., Oddy, V.H., Basarab, J.A., Cammack, K., Hayes, B J., Hegarty, R.S., McEwan, J.C., Miller, S., Pinares, C., and de Haas, Y. 2015. Invited review: Genetic possibilities to reduce enteric methane emissions from ruminants. Animal 9:1431-1440.</ref>).

=== Combining diet-based measurements with other proxies for methane emissions ===
Feed intake appears a reasonably adequate predictor of MeP: generally, heavier animals have higher maintenance requirements, so eat more and produce more CH4. However, a substantial level of variation is left unaccounted for. This suggests that more detailed information on dietary composition is needed. This is also important when one wants to account for MeP on diets of similar DMI but of different nutrient profiles.

The prediction accuracy of MeP strongly depends on the accuracy of quantifying the VFA produced in the rumen (Alemu et al., 2011<ref>Alemu, A.W., Dijkstra, J., Bannink, A., France, J., and Kebreab, E. 2011. Rumen stoichiometric models and their contribution and challenges in predicting enteric methane production. Anim. Feed Sci. Technol. 166-167:761-778.</ref>). The type of VFA formed during rumen fermentation depends on the type of substrate fermented (Bannink et al., 2011<ref>Bannink, A., van Schijndel, M.W., and Dijkstra, J. 2011. A model of enteric fermentation in dairy cows to estimate methane emission for the Dutch National Inventory Report using the IPCC Tier 3 approach. Anim. Feed Sci. Technol. 166-167:603-618.</ref>), such as the dietary content of neutral detergent fiber and starch. The type of substrate fermented thus appears a useful factor for predicting MeP (Ellis et al., 2007<ref>Ellis, J.L., Kebreab, E., Odongo, N.E., McBride, B.W., Okine, E.K., and France, J. 2007. Prediction of methane production from dairy and beef cattle. J. Dairy Sci. 90:3456–3466.</ref>), indicating that including a description of variation in dietary quality caused by nutritional factors results in improved prediction accuracy of CH4 emission (Ellis et al., 2010<ref>Ellis, J.L., Bannink, A., France, J., Kebreab, E., and Dijkstra, J. 2010. Evaluation of enteric methane prediction equations for dairy cows used in whole farm models. Glob. Change Biol. 16:3246–3256.</ref>; Moraes et al., 2014<ref name=":0">Moraes, L.E., Strathe, A.B., Fadel, J.G., Casper, D.P., and Kebreab, E. 2014. Prediction of enteric methane emissions from cattle. Glob. Change Biol. 20:2140–2148.</ref>).

=== Rumen ===
When feed intake is kept constant, a higher rumen capacity results in a lower passage rate (Demment and Van Soest, 1985<ref>Demment, M.W., and Van Soest, P.J. 1985. A nutritional explanation for body-size patterns of ruminant and nonruminant herbivores. Am. Nat. 125:641–672.</ref>), resulting in a higher MeP (Moraes et al., 2014<ref name=":0" />). Proxies based on rumen samples are generally poor to moderately accurate predictors of CH4, and are costly and difficult to measure routinely on-farm. VFA are a proxy for rumen CH4 emissions. Using rumen fermentation data obtained from in vitro gas production, Moss et al. (2000)<ref>Moss A.R., Jouany, J.P., and Newbold, J. 2000. Methane production by ruminants: Its contribution to global warming. Annal. Zootech. 49:231-253.</ref> reported a negative linear relationship between CH4 production and the ratio of (acetic + butyric acid)/propionic acid. However, by combining different information sources, either related to feed intake or to the impact of feed intake on the VFA composition, a better proxy with an improved accuracy can be achieved. This way, the prediction equation for CH4 production can be optimized (higher accuracy).

The relationship between rumen methanogen abundance and methanogenesis is less clear when changes in enteric CH4 emissions are modulated by diet or are a consequence of selecting phenotypes related to feed efficiency or MeY. Whereas in some reports there was a significant positive relationship (Aguinaga Casanas et al., 2015<ref name=":1">Aguinaga Casanas, M.A., , N., Krattenmacher, N., Thalle,r G., Metges, C.C., and Kuhla, B. 2015. Methyl-coenzyme M reductase A as an indicator to estimate methane production from dairy cows. J. Dairy Sci. 98:4074-4083.</ref>; Arndt et al., 2015<ref>Arndt, C., Powell, J.M., Aguerre, M.J., Crump, P.M., and Wattiaux, M.A. 2015. Feed conversion efficiency in dairy cows: Repeatability, variation in digestion and metabolism of energy and nitrogen, and ruminal methanogens. J. Dairy Sci. 98:3938-3950.</ref>; Sun et al., 2015<ref>Sun, X., Henderson, G., Cox, F., Molan,o G., Harrison, S.J., Luo, D., Janssen, P.H., and Pacheco, D. 2015. Lambs Fed Fresh Winter Forage Rape (Brassica napus L.) Emit Less Methane than Those Fed Perennial Ryegrass (Lolium perenne L.), and Possible Mechanisms behind the Difference. PLoS One 10(3):e0119697: DOI: 10.1371/journal.pone.0119697</ref>; Wallace et al., 2015<ref name=":2">Wallace, R., Rooke, J., McKain, N., Duthie, C.-A., Hyslop, J., Ross, D., Waterhouse, A., Watson, M., and Roehe, R. 2015. The rumen microbial metagenome associated with high methane production in cattle. BMC Genomics. 16:839.</ref>), in many others the concentration of methanogens was unrelated to methanogenesis (Morgavi et al., 2012<ref>Morgavi, D.P., Martin, C., Jouany, J.P., and Ranilla, M.J. 2012. Rumen protozoa and methanogenesis: not a simple cause-effect relationship. Br. J. Nutr. 107:388-397. 10.1017/S0007114511002935.</ref>; Kittelmann et al., 2014<ref>Kittelmann, S., Pinares-Patiño, C.S., Seedorf, H., Kirk, M.R., Ganesh, S., McEwan, J.C., and Janssen, P.H. 2014. Two different bacterial community types are linked with the low-methane emission trait in sheep. PLoS One 9:e103171.</ref>; Shi et al., 2014<ref name=":3">Shi W., Moon, C.D., Leahy, S.C., Kang, D., Froula, J., Kittelmann, S., Fan, C., Deutsch, S., Gagic, D., Seedorf, H., Kelly, W.J., Atua, R., Sang, C., Soni, P., Li, D., Pinares-Patiño, C.S., McEwan, J.C., Janssen, P.H., Chen, F., Visel, A., Wang, Z., Attwood, G.T., and Rubin, E.M. 2014. Methane yield phenotypes linked to differential gene expression in the sheep rumen microbiome. Genome Res. Doi:10.1101/gr.168245.113.</ref>; Bouchard et al., 2015<ref name=":4">Bouchard, K., Wittenberg, K.M., Legesse, G., Krause, D.O., Khafipour, E., Buckley, K.E., and Ominski, K.H. 2015. Comparison of feed intake, body weight gain, enteric methane emission and relative abundance of rumen microbes in steers fed sainfoin and lucerne silages under western Canadian conditions. Grass a Forage Sci. 70:116-129.</ref>). Bouchard et al. (2015)<ref name=":4" /> even reported a reduction in methanogens withoutsignificant decrease in MeP for steers fed sainfoin silage. Sheep selected for high or low MeY showed no differences in methanogen abundance, though there was a strong correlation with expression of archaeal genes involved in methanogenesis (Shi et al., 2014<ref name=":3" />).

Hindgut and Feces: whole tract digestibility variables cannot serve as primary predictors for enteric MeP in cattle or sheep, but might be used as supporting factors to improve the accuracy of prediction of CH4 output.

=== Protozoa and other rumen microbes ===
Protozoa are net producers of H2 and their absence from the rumen is associated with an average reduction in enteric MeP of approximately 11% (Hegarty, 1999<ref>Hegarty, R.S. 1999. Reducing rumen methane emissions through elimination of rumen protozoa. Aust. J. Agric. Res. 50:1321-1327.</ref>; Morgavi et al., 2010<ref>Morgavi, D.P., Forano, E., Martin, C., and Newbold, C.J. 2010. Microbial ecosystem and methanogenesis in ruminants. Animal 4:1024-1036.</ref>; Newbold et al., 2015<ref>Newbold, C.J., de la Fuente, G., Belanche, A., Ramos-Morales, E., and McEwan, N. 2015. The role of ciliate protozoa in the rumen. Front. Microbiol. 6:1313.doi: 10.3389/fmicb.2015.01313</ref>). Using a database of 28 experiments and 91 dietary treatments, Guyader et al. (2014)<ref>Guyader, J., Eugène, M., Nozière, P., Morgavi, D.P., Doreau, M., and Martin, C. 2014. Influence of rumen protozoa on methane emission in ruminants: a meta-analysis approach. Animal 8:1816-1825.</ref> showed a significant decrease of 8.14 g CH4/kg DMI for each log unit reduction in rumen protozoal abundance. About 21% of experiments within this dataset reported CH4 changes unrelated to protozoal abundance, highlighting the multifactorial nature of methanogenesis. Roehe et al. (2016)<ref>Roehe R., Dewhurst, R.J., Duthie, C-A., Rooke, J.A., McKain, N., Ross, D.W., Hyslop, J.J., Waterhouse, A., Freeman, T.C., Watson, M., and Wallace, R.J. 2016. Bovine Host Genetic Variation Influences Rumen Microbial Methane Production with Best Selection Criterion for Low Methane Emitting and Efficiently Feed Converting Hosts Based on Metagenomic Gene Abundance. PLoS Genet 12(2): e1005846. doi:10.1371/journal.pgen.1005846.</ref> observed that the ranking of sire groups for CH4 emissions measured with respiration chambers was the same as that for ranking on archaea/bacteria ratio, providing further evidence that host control of archaeal abundance contributes to genetic variation in CH4 emissions - at least in some circumstances. Across a wide geographical range, the methanogenic archaea were shown to be highly conserved across the world (Henderson et al., 2015<ref>Henderson, G., Cox, F., Ganesh, S., Jonker, A., Young, W., Global Rumen Census Collaborators, and Janssen, P.H. 2015. Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range. Sci. Rep. 5:14567. doi:10.1038/srep14567</ref>). This universality and limited diversity could make it possible to mitigate CH4 emissions by developing strategies that target the few dominant methanogens. However, one clear limitation of metagenomic predictions compared to genomic predictions was that the microbiome of the host is variable - that is, it may change in response to diet or other environmental factors over time, whereas the hosts DNA remains constant.

=== Rumen microbial genes ===
These included genes involved in the first and last steps of methanogenesis: formylmethanofuran dehydrogenase subunit B (fmdB) and methyl-coenzyme M reductase alpha subunit (mcrA), which were 170 times more abundant in high CH4 emitting cattle. Whilst gene-centric metagenomics is not low-cost or high-throughput, these results point to potential future proxy approaches using low-cost gene chips.

The difference in gene expression activity as opposed to abundance was also reported by others (Popova et al., 2011<ref>Popova, M., Martin, C., Eugène, M., Mialon, M.M., Doreau, M., and Morgavi, D.P. 2011. Effect of fibre- and starch-rich finishing diets on methanogenic Archaea diversity and activity in the rumen of feedlot bulls. Anim. Feed Sci. Technol. 166-167:113-121.</ref>). However, there are also studies in which there was no relationship with gene expression (Aguinaga Casanas et al., 2015<ref name=":1" />). There are some methodological and experimental differences that might explain some of the apparent contradictions, such as the type of gene target and primers used for nucleic acid amplification. Effects are seen most clearly when the difference in MeP between groups of animals is large (e.g. Wallace et al. (2015) <ref name=":2" />used treatments that generated a 1.9-fold difference CH4 emissions).

=== Proxies based on measurements in milk ===
Milk yield alone does not provide a good prediction of MeP by dairy cows. Yan et al. (2010)<ref>Yan. T., Mayne, C.S., Gordon, F.G., Porter, M.G., Agnew, R.E., Patterson, D.C., Ferris, C.P., and Kilpatrick, D.J. 2010. Mitigation of enteric methane emissions through improving efficiency of energy utilization and productivity in lactating dairy cows. J. Dairy Sci. 93:2630–2638. doi: 10.3168/jds.2009-2929.</ref> indicated that CH4 as a proportion of GE intake or milk energy output was negatively related to milk production. It is less clear if MeY can be predicted from milk yield when making comparisons across studies.

Milk MIR spectroscopy is relatively inexpensive, rapid and already routinely used technology in milk recording systems to predict fat, protein, lactose and urea contents in dairy milk to assist farm management decisions and breeding. It can be used as a promising strategy to exploit the link between enteric CH4 emission from ruminants and microbial digestion in the rumen by assessing the signature of digestion in milk composition. Milk MIR data can be obtained through regular milk recording schemes, as well as, on a herd level, through analysis used for milk payment systems. Diverse milk phenotypes can be obtained by MIR spectrometry – including detailed milk composition (e.g. FA as reported by Soyeurt et al., 2011<ref>Soyeurt H., Dehareng, F., Gengler, N., McParland, S., Wall, E., Berry, D.P., Coffey, M., and Dardenne, P. 2011. Mid-infrared prediction of bovine milk fatty acids across multiple breeds, production systems and countries. J. Dairy Sci. 94:1657–1667.</ref>), technological properties of milk, and cow physiological status (De Marchi et al., 2014<ref>De Marchi, M., Toffanin, V., Cassandro, M., and Penasa, M. 2014. Invited review: Mid-infrared spectroscopy as phenotyping tool for milk traits. J. Dairy Sci. 97:1171–1186. <nowiki>http://dx.doi.org/10.3168/jds.2013-6799</nowiki>.</ref>; Gengler et al., 2016<ref>Gengler, N., Soyeurt, H., Dehareng, F., Bastin, C., Colinet, F., Hammami, H., Vanrobays, M.-L., Lainé, A., Vanderick, S., Grelet, C., Vanlierde, A., Froidmont, E., and Dardenne, P. 2016. Capitalizing on fine milk composition for breeding and management of dairy cows. J. Dairy Sci. 99:4071-4079.</ref>). Several of these novel traits (i.e. FA composition) have been identified as potential indicators of CH4 emission. Therefore, using MIR to predict MeP (Dehareng et al. 2012<ref name=":5">Dehareng, F., Delfosse, C., Froidmont, E., Soyeurt, H., Martin, C., Gengler, N., Vanlierde, A., and Dardenne, P. 2012. Potential use of milk mid-infrared spectra to predict individual methane emission of dairy cows. Animal 6:1694-701.</ref>; Vanlierde et al. 2013<ref>Vanlierde, A., Dehareng, F., Froidmont, E., Dardenne, P., Kandel, P.B., Gengler, N., Deighton, M.H., buckley, F., Lewis, E., McParland, S., Berry, D.P., and Soyeurt, H. 2013. Prediction of the individual enteric methane emission of dairy cows from milk-mid-infrared spectra. Advances in Animal Biosciences. 5th Greenhouse Gases Animal Agriculture Conference (GGAA2013) 23-26 June 2014. Dublin, Ireland. p 433.</ref>, 2015<ref name=":6">Vanlierde, A., Vanrobays, M.L., Dehareng, F., Froidmont, E., Soyeurt, H., McParland, S., Lewis, E., Deighton, M.H., Grandl, F., Kreuzer, M., Grendler, B., Dardenne, P., and Gengler, N. 2015. Hot topic: Innovative lactation-stage-dependent prediction of methane emissions from milk mid-infrared spectra. J. Dairy Sci. 98:5740–5747.</ref>; Van Gastelen and Dijkstra, 2016<ref name=":7">Van Gastelen, S., and Dijkstra, J.. 2016. Prediction of methane emission from lactating dairy cows using milk fatty acids and mid-infrared spectroscopy. J. Sci. Food Agric. 96:3963-3968. DOI: 10.1002/jsfa.7718.</ref>) is also a logical extension of its use to quantify the major milk components (i.e. fat, protein, casein, lactose, and urea) and minor components (e.g. FA). Dehareng et al. (2012)<ref name=":5" /> assessed the feasibility to predict individual MeP from dairy cows using milk MIR spectra. Their initial results suggest that this approach could be useful to predict MeP at the farm or regional scale, as well as to identify low-CH4 emitting cows. According to Van Gastelen and Dijkstra (2016)<ref name=":7" />, MIR spectroscopy has the disadvantage that it has a moderate predictive power for CH4 emission, both direct and indirect (i.e. via milk FA), and that it lacks the ability to predict important milk FA for CH4 prediction. They concluded that it may not be sufficient to predict MeP based on MIR alone. It is, however, possible to improve the accuracy of prediction through the combination of MIR with some animal characteristics such as lactation stage (Vanlierde et al., 2015<ref name=":6" />). The advantage of this latter development is that this type of prediction can be done on a very large scale inside a routine milk recording system (Vanlierde et al., 2015<ref name=":6" />).

=== Proxy: future developments and perspectives ===
There is currently limited consensus on which phenotype to use to lower the carbon footprint of milk production through genetic selection. This could be MeP, MeI or MeY. The direct goal would be CH4 production; the relationship with milk production and/or feed intake could be accounted for by including these in the final selection index or scheme. However, one might argue that it would be more effective/accurate to directly use milk production- or feed intakecorrected CH4 (e.g. CH4 intensity or yield) as breeding goal.

The analysis of proxies in terms of their attributes shows that proxies that are based on samples from the rumen or related to rumen sources are poor to moderately accurate predictors of CH4. In addition, these proxies are too costly and difficult for routine on-farm implementation. On the other hand, proxies related to BW, milk yield and composition (e.g. milk FA) are moderately to highly accurate predictors of CH4 and relatively simple, low-cost and easier to implement in practice (Cassandro et al.,2010<ref>Cassandro, M., Cecchinato, A., Battagin, M., Penasa, M., 2010. Genetic parameters of predicted methane production in Holstein Friesian cowsIn: Proc. 9th World Congr. on Genetics Applied to Livestock Production, Leipzig, Germany. . Page 181</ref>; Cassandro, 2013<ref>Cassandro, M. 2013. Comparing local and cosmopolitan cattle breeds on added values for milk and cheese production and their predicted methane emissions. Animal Genetic Resources/Ressources génétiques animales/Recursos genéticos animales, available on CJO2013. doi:10.1017/S2078 63361200077X. </ref>). Particularly, milk MIR and the prediction of CH4 based on milk MIR along with other covariates such as lactation stage is a promising alternative: that is accurate, cheaper and easy to be implemented in routine milk analysis at no extra cost.

Therefore, in the future advances in infrared, photoacoustic and related technologies will push the boundaries, particularly in focusing on developments of fast and portable technologies. Such developments will lead to better and promising proxies for CH4 that will enable a sizable throughput of CH4 phenotypes in dairy cows. Antunes-Fernandes et al. (2016)<ref>Antunes-Fernandes, E.C., van Gastelen, S., Dijkstra, J., Hettinga K.A., and Vervoort, J. 2016. Milk metabolome relates enteric methane emission to milk synthesis, and energy metabolism pathways. J. Dairy. Sci. 99:6251-6262.</ref> already presented the use of metabolomics on milk to better understand the biological pathways involved in CH4 production in dairy cattle. The techniques used in that study are not suitable for large scale measurements, but rapid developments in omics may offer tests and assay methodologies on blood, urine or milk samples that will provide an additional tool for developing new / additional proxies for CH4 emissions in dairy cattle.

Section 20: Proxies

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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]].

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== Introduction ==
Large-scale measurements of enteric CH4 emissions from dairy cows are needed for effective monitoring of strategies to reduce the carbon footprint of milk production, as well as for incorporation of CH4 emissions into breeding programs. However, measurements on a sufficiently large scale are difficult and expensive. Proxies for CH4 emissions can provide an alternative, but each approach has limitations. Negussie et al. (2019)<ref>Negussie, E., González Recio, O., de Haas, Y., Gengler N., Soyeurt, H., Peiren, N., Pszczola, M., Garnsworthy, P., Battagin, M., Bayat, A., Lassen, J., Yan, T., Boland, T., Kuhla, B., Strabel, T., Schwarm, A., Vanlierde, A., and Biscarini, F. 2019. Machine learning ensemble algorithms in predictive analytics of dairy cattle methane emission using imputed versus non-imputed datasets. 7th GGAA – Greenhouse Gas and Animal Agriculture Conference held from August 4th to 8th, Iguassu Falls/Brazil. Oral communication, Book of Abstracts Page 40. <nowiki>http://www.ggaa2019.org/sites/default/files/proceedings-ggaa2019.pdf</nowiki></ref> recently showed the potential of proxies proxies that are easy to record in the farm. These proxies can be gathered in most farms and are a realistic threshold accuracy that can be obtained without more fancy proxies. Several techniques have been developed for the measurement of CH4 emissions from ruminants, with varying degrees of accuracy (see reviews by Cassandro et al., 2013<ref>Cassandro, M., Mele, M., Stefanon, B.. 2013. Genetic aspects of enteric methane emission in livestock ruminants. Italian J. Anim. Sci. 12:e73: 450-458.</ref> and Hammond et al., 2016A<ref>Hammond, K.J., Crompton, L.A., Bannink, A., Dijkstra, J., Yáñez-Ruiz, D.R., O’Kiely, P., Kebreab, E., Eugenè, M.A., Yu, Z., Shingfield, K.J., Schwarm, A., Hristov, A.N., and Reynolds, C.K. 2016A. Review of current in vivo measurement techniques for quantifying enteric methane emission from ruminants. Anim. Feed Sci. Technol. 219:13–30. doi:10.1016/j.anifeedsci.2016.05.018.</ref>), but routine individual measurements on a large scale (a requisite for genetic selection) have proven to be difficult and expensive (Pickering et al., 2015<ref>Pickering, N.K., Oddy, V.H., Basarab, J.A., Cammack, K., Hayes, B J., Hegarty, R.S., McEwan, J.C., Miller, S., Pinares, C., and de Haas, Y. 2015. Invited review: Genetic possibilities to reduce enteric methane emissions from ruminants. Animal 9:1431-1440.</ref>; Negussie et al., 2016<ref>Negussie E., Lehtinen, J., Mäntysaari, P., Liinamo, A-E., Mäntysaari, E., and Lidauer, M.. 2016. Non-invasive individual methane measurements in dairy cows using photoacoustic infrared spectroscopy technique. 6th Greenhouse Gases Animal Agriculture Conference (GGAA2016) 14-18 February 2016. Melbourne, Australia. Abstract. p62.</ref>). Therefore, identifying proxies (i.e. indicators or indirect traits) that are correlated to CH4 emission, but which are easy and relatively low-cost to record on a large scale, is a much needed alternative. Proxies might be less accurate, but could be measured repeatedly to reduce random noise. The (potential) proxies range from simple and low-cost measurements such as body weight, to high-throughput milk MIR, to more demanding measures like rumen morphology, rumen metabolites or microbiome profiling.

Combining proxies that are easy to measure and cheap to record could provide predictions of CH4 emissions that are sufficiently accurate for selection and management of cows with low CH4 emissions.

== Available Proxies ==
A large array of CH4 proxies differing widely in accuracy and applicability under different conditions have been reported. The ideal proxy would be highly phenotypically and genetically correlated with CH4 emissions and could easily, and potentially repeatedly, be measured on a large scale. A systematic summary and assessment of existing knowledge is needed for the identification of robust and accurate CH4 proxies for future use. Table 5 summarizes proxies for CH4 production, and Table 6 summarizes results from combining proxies to improve predictability of proxies for CH4 prediction.
{| class="wikitable"
|+
!Proxy
!Description / conclusion
!Reference
|-
| colspan="3" |'''(1) Feed intake and feeding behavior'''
|-
|Dry Matter Intake (DMI)
|DMI predict MeP with R2= 0.06-0.64, and ME intake predict MeP with R2= 0.53-0,55
| Ellis et al. (2007);<ref>Ellis, J.L., Kebreab, E., Odongo, N.E., McBride, B.W., Okine, E.K., and France, J. 2007. Prediction of methane production from dairy and beef cattle. J. Dairy Sci. 90:3456–3466.</ref>
Mills et al. (2003)<ref>Mills, J.A.N., Kebreab, E., Yates, C.M., Crompton, L.A., Cammell, S.B., Dhanoa, M.S., Agnew, R.E., and France, J. 2003. Alternative approaches to predicting methane emissions from dairy cows. J. Anim. Sci. 81:3141–3150.</ref>;

Negussie et al. (2019)<ref>Negussie, E., González Recio, O., de Haas, Y., Gengler N., Soyeurt, H., Peiren, N., Pszczola, M., Garnsworthy, P., Battagin, M., Bayat, A., Lassen, J., Yan, T., Boland, T., Kuhla, B., Strabel, T., Schwarm, A., Vanlierde, A., and Biscarini, F. 2019. Machine learning ensemble algorithms in predictive analytics of dairy cattle methane emission using imputed versus non-imputed datasets. 7th GGAA – Greenhouse Gas and Animal Agriculture Conference held from August 4th to 8th, Iguassu Falls/Brazil. Oral communication, Book of Abstracts Page 40. <nowiki>http://www.ggaa2019.org/sites/default/files/proceedings-ggaa2019.pdf</nowiki></ref>
|-
|Gross Energy intake (GE)
|Predict MeP with RMSPE= 3.01.
| Moraes et al. (2014)<ref name=":0">Moraes, L.E., Strathe, A.B., Fadel, J.G., Casper, D.P., and Kebreab, E. 2014. Prediction of enteric methane emissions from cattle. Glob. Change Biol. 20:2140–2148.</ref>
|-
|Feeding behavior
| Magnitude and direction of relation to MeP varies across studies
|Nkrumah et al. (2006);<ref>Nkrumah, J.D., Okine, E.K., Mathison, G.W., Schmid, K., Li, C., Basarab, J.A., Price, M.A., Wang, Z., and Moore, S.S. 2006. Relationships of feedlot feed efficiency, performance, and feeding behavior with metabolic rate, methane production, and energy partitioning in beef cattle. J. Anim. Sci. 84:145-153.</ref>
Jonker et al., 2014<ref>Jonker, A., Molano, G., Antwi, C., Waghorn, G.. 2014. Feeding lucerne silage to beef cattle at three allowances and four feeding frequencies affects circadian patterns of methane emissions, but not emissions per unit of intake. Anim. Prod. Sci.54:1350-1353.</ref>
|-
| Rumination time
|High rumination relates to more milk, consume more concentrate and produce more CH4, lower RMP and MeI
| Watt et al. (2015)<ref>Watt, L.J., Clark, C.E.F., Krebs, G.L., Petzel, C.E., Nielsen, S., and Utsumi, S.A. 2015. Differential rumination, intake, and enteric methane production of dairy cows in a pasture-based automatic milking system. J. Dairy Sci. 98:7248–7263.</ref>;
López- Paredes et al. (2020)<ref>Lopez-Paredes, J., Goiri, I., Atxaerandio, R., García-Rodríguez, A., Ugarte, E., Jiménez-Montero, J.A., Alenda, R and González-Recio, O. 2020. Mitigation of greenhouse gases in dairy cattle via genetic selection (i): Genetic parameters of direct methane using non-invasive methods and its proxies. J. Dairy Sci. 103.</ref>
|-
|Rumen microbiome
|The metagenome can predict DMI, and classify high vs low intakes
|Delgado et al. (2019)<ref>Delgado, B., Bach A., Guasch I., González C, Elcoso G., Pryce J.E., Gonzalez-Recio O. (2019).Whole rumen metagenome sequencing allows classifying and predicting feed efficiency and intake levels in cattle. Scientific Reports 9: 11. doi:10.1038/s41598-018-36673-w</ref>
|-
| colspan="3" |'''(2) Rumen function, metabolites and microbiome'''
|-
|Dietary antimethanogenic compounds
|Inhibitors of the enzyme methyl coenzyme-M reductase: bromochoromethane; chloroform; 3- nitrooxypropanol (not always)
|Denman et al., 2007;<ref>Denman, S.E., Tomkins, N.W., and McSweeney, C.S. 2007. Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane. FEMS Microbiol. Ecol. 62:313-322.</ref>
Knight et al., 2011;<ref>Knight, T., Ronimus, R.S., Dey, D., Tootill, C., Naylor, G., Evans, P., Molano, G., Smith, A., Tavendale, M., Pinares-Patiño, C.S., and Clark, H. 2011. Chloroform decreases rumen methanogenesis and methanogen populations without altering rumen function in cattle. Anim. Feed Sci. Technol. 166-167:101-112.</ref>

Haisan et al., 2014<ref>Haisan, J., Sun, Y., Guan, L.L., Beauchemin, K.A., Iwaasa, A., Duval, S., Barreda, D.R., and Oba, M. 2014. The effects of feeding 3-nitrooxypropanol on methane emissions and productivity of Holstein cows in mid lactation. J. Dairy Sci. 97:3110-3119.</ref>;

Romero-Perez et al., 2014<ref>Romero-Perez, A., Okine, E.K., McGinn, S.M., Guan, L.L., Oba, M., Duval, S.M., Kinderman,n M., and Beauchemin, K.A. 2014. The potential of 3-nitrooxypropanol to lower enteric methane emissions from beef cattle. J. Anim. Sci. 92:4682-4693.</ref>
|-
|Dietary antimicrobial compounds
|Induce reductions in both MeP and methanogens numbers: nitrates, anacardic acid (cashew nut shell liquid), monensin, isobutyrate
|Iwamoto et al., 2002;<ref>Iwamoto, M., Asanuma, N., and Hin,o T. 2002. Ability of Selenomonas ruminantium, Veillonella parvula, and Wolinella succinogenes to reduce nitrate and nitrite with special reference to the suppression of ruminal methanogenesis. Anaerobe. 8:209-215.</ref>
Kubo et al., 1993<ref>Kubo, I., Muroi, H., Himejima, M., Yamagiwa, Y., Mera, H., Tokushima, K., Ohta, S., and Kamikawa, T. 1993. Structure-antibacterial activity relationships of anacardic acids. J. Agric. Food Chem. 41:1016-1019.</ref>;

van Zijderveld et al., 2010<ref>Van Zijderveld, S.M., Gerrits, W.J.J., Apajalahti, J.A., Newbold, J.R., Dijkstra, J., Leng, R A., and Perdok, H.B. 2010. Nitrate and sulfate: effective alternative hydrogen sinks for mitigation of ruminal methane production in sheep. J. Dairy Sci. 93:5856-5866.</ref>;

Veneman et al., 2015<ref>Veneman, J.B., Muetzel, S., Hart, K.J., Faulkner, C.L., Moorby, J.M., Perdok, H.B., and Newbold, C.J. 2015. Does Dietary Mitigation of Enteric Methane Production Affect Rumen Function and Animal Productivity in Dairy Cows? PLoS ONE 10(10): e0140282. doi: 10.1371/journal.pone.0140282</ref>;

Shinkai et al., 2012<ref>Shinkai, T., Enishi, O., Mitsumori, M., Higuchi, K., Kobayashi, Y., Takenaka, A., Nagashima, K., and Mochizuki, M. 2012. Mitigation of methane production from cattle by feeding cashew nut shell liquid. J. Dairy Sci. 95:5308-5316.</ref>;

Wang et al., 2015<ref>Wang, C., Liu, Q., Zhang, Y.L., Pei, C.X., Zhang, S.L., Wang, Y.X., Yang, W.Z., Bai, Y.S., Shi, Z.G., and Liu, X.N. 2015. Effects of isobutyrate supplementation on ruminal microflora, rumen enzyme activities and methane emissions in Simmental steers. J. Anim. Physiol. Anim. Nutr. (Berl). 99:123-131.</ref>
|-
|Rumen microbiome profile
|High Fibrobacteres, Quinella ovalis and Veillonellaceae and low Ruminococcaceae, Lachnospiraceae and Clostridiales associate with low- CH4 phenotypes and high propionate
Protozoa concentration
|Kittelmann et al., 2014<ref>Kittelmann, S., Pinares-Patiño, C.S., Seedorf, H., Kirk, M.R., Ganesh, S., McEwan, J.C., and Janssen, P.H. 2014. Two different bacterial community types are linked with the low-methane emission trait in sheep. PLoS One 9:e103171.</ref>;
Wallace et al., 2014<ref>Wallace, R. ., Rooke, J A., Duthie, C.-A., Hyslop, J.J., Ross, D.W., McKain, N., de Souza, S.M., Snelling, T.J., Waterhouse, A., and Roehe, R. 2014. Archaeal abundance in post-mortem ruminal digesta may help predict methane emissions from beef cattle. Sci. Rep. 4:5892.</ref>;

Sun et al., 2015<ref>Sun, X., Henderson, G., Cox, F., Molan,o G., Harrison, S.J., Luo, D., Janssen, P.H., and Pacheco, D. 2015. Lambs Fed Fresh Winter Forage Rape (Brassica napus L.) Emit Less Methane than Those Fed Perennial Ryegrass (Lolium perenne L.), and Possible Mechanisms behind the Difference. PLoS One 10(3):e0119697: DOI: 10.1371/journal.pone.0119697</ref>;

Guyader et al., 2014<ref>Guyader, J., Eugène, M., Nozière, P., Morgavi, D.P., Doreau, M., and Martin, C. 2014. Influence of rumen protozoa on methane emission in ruminants: a meta-analysis approach. Animal 8:1816-1825.</ref>
|-
|Rumen microbiome profile
|Predict MeP with R2 up to 0.55
|Ross et al. 2013a<ref>Ross, E. M., P.J . Moate, L.C. Marett, B.G. Cocks and B.J. Hayes. 2013a. Investigating the effect of two methane-mitigating diets on the rumen microbiome using massively parallel sequencing. J. Dairy Sci. 96:6030–6046.</ref>;
Ross et al. (2013b)<ref>Ross, E. M., Moate, P. J., Marett, L.C., Cocks, B.G., and Hayes, B.J. 2013b. Metagenomic Predictions: From Microbiome to Complex Health and Environmental Phenotypes in Humans and Cattle. PLoS One DOI: 10.1371/journal.pone.0073056.</ref>
|-
|Microbial genes
|20 (out of 3970 identified) related to CH4 emissions
|Roehe et al. (2016)<ref>Roehe R., Dewhurst, R.J., Duthie, C-A., Rooke, J.A., McKain, N., Ross, D.W., Hyslop, J.J., Waterhouse, A., Freeman, T.C., Watson, M., and Wallace, R.J. 2016. Bovine Host Genetic Variation Influences Rumen Microbial Methane Production with Best Selection Criterion for Low Methane Emitting and Efficiently Feed Converting Hosts Based on Metagenomic Gene Abundance. PLoS Genet 12(2): e1005846. doi:10.1371/journal.pgen.1005846.</ref>
|-
|Rumen volume (Xray Computed Tomography) and retention time
|Low-MeY sheep had smaller rumens. Faster passage= less time to ferment substrate - explained 28% of variation in MeP
|Pinares Patiño et al., 2003<ref>Pinares-Patiño C.S., Hickey, S.M., Young, E.A., Dodds, K.G., MacLean, S., Molano, G., Sandoval, E., Kjestrup, H., Harland, R., Pickering, N.K., and McEwan, J.C. 2013. Heritability estimates of methane emissions from sheep. Animal 7: 316–321.</ref>;
Goopy et al., 2014<ref>Goopy, J.P., Donaldson, A., Hegarty, R., Vercoe, P.E., Haynes, F., Barnett, M., and Oddy, V.H. 2014. Low-methane yield sheep have smaller rumens and shorter rumen retention time. Br. J. Nutr. 111:578-585.</ref>;

Okine et al. (1989)<ref>Okine, E. ., Mathiso,n G.W., and Hardin, R.T. 1989. Effects of changes in frequency of reticular contractions on fluid and particulate passage rates in cattle. J. Anim. Sci. 67:3388–3396.</ref>
|-
|Blood triiodothyronine concentration
|Reduced MeY
|Barnett et al. (2012)<ref>Barnett, M.C., Goopy, J.P., McFarlane, J.R., Godwin, I.R., Nolan, J.V., and Hegarty, R.S. (2012). Triiodothyronine influences digesta kinetics and methane yield in sheep. Anim. Prod. Sci. 52:572-577.</ref>
|-
|Acetate to propionate ratio in ruminal fluid
|Positively associated with CH4 emissions, but not confirmed in all studies, sometimes opposite relation
|Mohammed et al., 2011;<ref name=":1">Mohammed, R., McGinn, S.M., and Beauchemin, K.A. 2011. Prediction of enteric methane output from milk fatty acid concentrations and rumen fermentation parameters in dairy cows fed sunflower, flax, or canola seeds. J. Dairy Sci. 94:6057–6068.</ref>
Fievez et al., 2012<ref>Fievez V., Colma,n E., Castro-Montoya, J.M., Stefanov, I., and Vlaeminck, B. 2012. Milk odd- and branched-chain fatty acids as biomarkers of rumen function – An update. Anim. Feed. Sci. Technol. 172:51–65.</ref>;

Chung et al., 2011<ref>Chung, Y.-H., Walker, N.D., McGinn, S.M., and Beauchemin, K.A. 2011. Differing effects of 2 active dried yeast (Saccharomyces cerevisiae) strains on ruminal acidosis and methane production in nonlactating dairy cows. J. Dairy Sci. 94:2431–2439.</ref>;

Van Zijderveld et al., 2010<ref>Van Zijderveld, S.M., Gerrits, W.J.J., Apajalahti, J.A., Newbold, J.R., Dijkstra, J., Leng, R A., and Perdok, H.B. 2010. Nitrate and sulfate: effective alternative hydrogen sinks for mitigation of ruminal methane production in sheep. J. Dairy Sci. 93:5856-5866.</ref>
|-
| colspan="3" |'''(3) Milk production and composition'''
|-
|Modelling approach
|Doubling milk production only adds 5 kg to the MeP and so greatly reduces MeY
|Kirchgessner et al. (1995)<ref>Kirchgessner M., Windisch, W., and Muller, H.L. 1995. Nutritional factors for the quantification of methane production. In: Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction. Proceedings 8th International Symposium on Ruminant Physiology (eds W. Von Engelhardt, S. Leonhard-Marek, G. Breves and D. Giesecke). Reproduction Proceedings 8th International Symposium on Ruminant Physiology. Ferdinand Enke Verlag, Stuttgart, Germany. pp. 333-348.</ref>;
Hristov et al. (2014)<ref>Hristov, A.N., Johnson, K.A., and Kebreab, E, 2014. Livestock methane emissions in the United States. Proc. Natl. Aacad. Sci. 111:E1320; doi:10.1073/pnas.1401046111</ref>
|-
|Milk fat content
|key explanatory variable for predicting CH4: A moderate negative genetic correlation with infrared predicted
MeI: correlations MeP = 0,08 and MeI = - 0.13
|Moraes et al. (2014)<ref>Moraes, L.E., Strathe, A.B., Fadel, J.G., Casper, D.P., and Kebreab, E. 2014. Prediction of enteric methane emissions from cattle. Glob. Change Biol. 20:2140–2148.</ref>;
Kandel et al., 2014A<ref name=":2">Kandel, P.B., Vanderick, S., Vanrobays, M.L., Vanlierde, A., Dehareng, F., Froidmont, E., Soyeurt, H., and Gengler, N. 2014A. Consequences of selection for environmental impact traits in dairy cows. Page 19. (<nowiki>http://orbi.ulg.ac.be/bitstream/2268/164402/164401/NSABS162014_poster_Purna_abstract.pdf</nowiki>) I:n Proc. 19th National symposium on applied biological sciences, Gembloux, Belgium.</ref>, B<ref name=":3">Kandel, P.B., Vanderick, S., Vanrobays, M.L., Vanlierde, A., Dehareng, F., Froidmont, E., Soyeur,t H., and Gengler, N. 2014B. Consequences of selection for environmental impact traits in dairy cows. In: 10th World Congress on Genetics Applied to Livestock Production (WCGALP), 17-22 August, 2014. Vancouver, Canada.</ref>;

Vanlierde et al. (2015)<ref name=":4">Vanlierde, A., Vanrobays, M.L., Dehareng, F., Froidmont, E., Soyeurt, H., McParland, S., Lewis, E., Deighton, M.H., Grandl, F., Kreuzer, M., Grendler, B., Dardenne, P., and Gengler, N. 2015. Hot topic: Innovative lactation-stage-dependent prediction of methane emissions from milk mid-infrared spectra. J. Dairy Sci. 98:5740–5747.</ref>
|-
|Milk fat content
|A positive relationship between VFA proportions and methanogenesis is expected as a consequence of the common biochemical pathways; Dietary unsaturated fatty acids are negatively associated with CH4 emissions
|Vlaeminck et al., 2006<ref>Vlaeminck, B., Fievez, V., Cabrita, A.R.J., Fonseca, A.J.M., and Dewhurst, R.J. 2006. Factors affecting odd- and branched-chain fatty acids in milk: A review. Anim. Feed Sci. Technol. 131:389–417.</ref>;
Van Lingen et al., 2014<ref name=":5">Van Lingen H.J., Crompton, L.A., Hendriks, W.H., Reynolds, C.K., Dijkstra, J. 2014. Meta-analysis of relationships between enteric methane yield and milk fatty acid profile in dairy cattle. J. Dairy Sci. 97:7115-7132.</ref>
|-
|Milk protein yield
|Correlation with Mel = - 0.47 or -0.09, MeP = 0.53
|Kandel et al. (2014)<ref name=":2" />;
Vanlierde et al. (2015)<ref name=":4" />
|-
|Lactose
|Variable correlations: MeP = 0,33; MeI = - 0.21; R = 0.19 for CH4 emission
|Miettinen and Huhtanen (1996)<ref>Miettinen, H., and Huhtanen, P. 1996. Effects of the ration of ruminal propionate to butyrate on milk yield and blood metabolites in dairy cows. J. Dairy Sci. 79:851–861.</ref>;
Dehareng et al. (2012)<ref>Dehareng, F., Delfosse, C., Froidmont, E., Soyeurt, H., Martin, C., Gengler, N., Vanlierde, A., and Dardenne, P. 2012. Potential use of milk mid-infrared spectra to predict individual methane emission of dairy cows. Animal 6:1694-701.</ref>
|-
|Somatic cell score
|Genetic correlation with infrared predicted MeI: R = 0.07
|Kandel et al. (2014A<ref name=":2" />, B<ref name=":3" />)
|-
|Prediction equations Milk FA and CH4 emissions, including from MIR data
|R² ranged between 47 and 95%; relationships between the individual milk FA and MeP differed considerably and the correlations between CH4 and milk FA vary throughout the lactation
|Chilliard et al. (2009)<ref>Chilliard Y., Martin ,C., Rouel, J., and Doreau, M. 2009. Milk fatty acids in dairy cows fed whole crude linseed, extruded linseed, or linseed oil, and their relationship with methane output. J. Dairy Sci. 92:5199-5211.</ref>;
Delfosse et al. (2010)<ref>Delfosse, O., Froidmont, E., Fernandez Pierna, J. A., Martin, C., and Dehareng, F. 2010. Estimation of methane emissions by dairy cows on the basis of milk composition. In: Greenhouse Gases and Animal Agriculture Conference. 2010; GGAA2010: 4. Greenhouse Gases and Animal Agriculture Conference, Banff, CAN, 2010-10-03-2010-10-08, 60-61.</ref>;

Castro-Montoya et al. (2011)<ref>Castro Montoya, J., Bhagwat, A.M., Peiren, N., De Campeneere, S., De Baets, B., and Fievez, V. 2011. Relationships between odd- and branched-chain fatty acid profiles in milk and calculated enteric methane proportion for lactating dairy cattle. Anim. Feed Sci. Technol.166:596–602.</ref>;

Dijkstra et al. (2011)<ref>Dijkstra, J., van Zijderveld, S.M., Apajalahti, J.A., Bannink, A., Gerrits, W.J.J., Newbold, J.R., Perdok, H.B.,, and Berends, H. 2011. Relationships between methane production and milk fatty acid profiles in dairy cattle. Anim. Feed Sci. Technol. 166–167:590–595.</ref>;

Kandel et al. (2013)<ref>Kandel, P.B., Vanrobays, M.L., Vanlierde, A., Dehareng, F., Froidmont, E., Dardenne, P., Lewis, E., Buckley, F., Deighton, M.H., McParland, S. and Gengler, N., 2013. Genetic parameters for methane emissions predicted from milk mid-infrared spectra in dairy cows. J. Dairy Sci. 95(E-1):p.388.</ref>;

Mohammed et al. (2011)<ref name=":1" />;

Van Lingen et al. (2014)<ref name=":5" />;

Williams et al. (2014)<ref>Williams, S.R.O., Williams, B., Moate, P.J., Deighton, M.H., Hannah, M.C., and Wales, W.J. 2014. Methane emissions of dairy cows cannot be predicted by the concentrations of C8:0 and total C18 fatty acids in milk. Anim. Prod. Sci. 54:1757–1761. <nowiki>http://dx.doi.org/10.1071/AN14292</nowiki>.</ref>;

Dijkstra et al. (2016)<ref>Dijkstra, J., van Zijderveld, S.M., Apajalahti, J.A., Bannink, A., Gerrits, W.J.J., Newbold, J.R., Perdok, H.B.,, and Berends, H. 2011. Relationships between methane production and milk fatty acid profiles in dairy cattle. Anim. Feed Sci. Technol. 166–167:590–595.</ref>;

Rico et al. (2016)<ref>Rico D.E., Chouinard, P.Y., Hassanat, F., Benchaar, C., and Gervais, R. 2016. Prediction of enteric methane emissions from Holstein dairy cows fed various forage sources. Animal 10:203-211. doi:10.1017/S1751731115001949</ref>;

Van Gastelen and Dijkstra (2016)<ref>Van Gastelen, S., and Dijkstra, J.. 2016. Prediction of methane emission from lactating dairy cows using milk fatty acids and mid-infrared spectroscopy. J. Sci. Food Agric. 96:3963-3968. DOI: 10.1002/jsfa.7718.</ref>;

Vanrobays et al. (2016);<ref>Vanrobays, M.-L., Bastin, C., Vandenplas, J., Hammami, H., Soyeurt, H., Vanlierde, A., Dehareng, F., Froidmont, E., and Gengler, N. 2016. Changes throughout lactation in phenotypic and genetic correlations between methane emissions and milk fatty acid contents predicted from milk mid-infrared spectra. J. Dairy Sci. 99:1–14. <nowiki>http://dx.doi.org/10.3168/jds.2015-10646</nowiki></ref>

Bougoin et al., (2019)<ref>Bougouin, A., Appuhamy, J.A.D.R.N., Ferlay, A., Kebreab, E., Martin, C., Moate, P.J., Benchaar, C., Lund, P., and Eugène, M. 2019. Individual milk fatty acids are potential predictors of enteric methane emissions from dairy cows fed a wide range of diets: Approach by meta-analysis. J. Dairy Sci. 102:10616–10631. DOI: <nowiki>http://doi.org/10.3168/jds.2018-15940</nowiki></ref>
|-
| colspan="3" |'''(4) Hind-gut and feces'''
|-
|Whole tract digestibility (potential as supporting factors in the prediction of enteric CH4 emissions)
|Main effects relate to rumen (see above), but energy digestibility as a supporting factor to GE intake improved the accuracy of CH4 prediction, despite the fact that there was no direct linear relationship between energy digestibility and MeY and in % of GE intake
|Yan et al., 2009 C<ref name=":6">Yan, T., Porter, M.G., and Mayne, S.C. 2009. Prediction of methane emission from beef cattle using data measured in indirect open-circuit respiration calorimeters. Animal 3:1455-1462.</ref>
|-
|Ratio of acetic and butyric acid divided by propionic acid
|Methane yield positive relation
|Moss et al., 2000<ref>Moss A.R., Jouany, J.P., and Newbold, J. 2000. Methane production by ruminants: Its contribution to global warming. Annal. Zootech. 49:231-253.</ref>
|-
| colspan="3" |'''(5) Whole animal measurements'''
|-
|Body weight and conformation
|Prediction models; primary predictor for enteric MeP
|Moraes et al. (2014)<ref name=":0" />;
Holter and Young, 1992; <ref>Holter J.B., and Young, A.J. 1992. Methane production in dry and lactating Holstein cows. J. Dairy Sci. 75:2165–2175.</ref>

Yan et al., 2009<ref name=":6" />
|-
|Body weight
|Relationship with MeI: r = 0.44; relationship between body weight and rumen capacity
|Antunes-Fernandes et al. (2016)<ref>Antunes-Fernandes, E.C., van Gastelen, S., Dijkstra, J., Hettinga K.A., and Vervoort, J. 2016. Milk metabolome relates enteric methane emission to milk synthesis, and energy metabolism pathways. J. Dairy. Sci. 99:6251-6262.</ref>;
Demment and Van Soest, 1985<ref>Demment, M.W., and Van Soest, P.J. 1985. A nutritional explanation for body-size patterns of ruminant and nonruminant herbivores. Am. Nat. 125:641–672.</ref>
|-
|Body weight
|Key explanatory variable for enteric MeP
|No reference available
|-
|Conformation traits: affects enteric MeP
|Indicators for rumen volume (via feed intake and rumen passage rates); BCS
|Agnew and Yan, 2000<ref>Agnew, R.E. and Yan, T. 2000. The impact of recent research on energy feeding systems for dairy cattle. Livest. Prod. Sci. 66:197-215.</ref>
|-
|Lactation stage
|Complementary proxy
|Vanlierde et al. (2015)<ref name=":4" />
|}Table 5. Available methane proxies include: (1) feed intake and feeding behaviour, (2) rumen function, metabolites and microbiome, (3) milk production and composition, (4) hind-gut and faeces, and (5) measurements at the level of the whole animal. It is evident that no single proxy offers a good solution in terms of all of these attributes, though the low cost and high throughput make milk MIR a good candidate for further work on refining methods, improving calibrations and exploring combinations with other proxies.

Section 20: Pro's and con's of devices

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Bgolden:

Section 20: Correlations among methods

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Bgolden: