An investigation conducted in Brazil analyzed all the microorganisms present in the feces of Nelore bulls and found biomarkers that can identify animals with a low-emission, feed-efficient phenotype (photo: Juliana Sussai/EMBRAPA Pecuária Sudeste)
Published on 11/07/2022
By Karina Ninni | Agência FAPESP – By analyzing the microorganisms present in beef cattle stool (feces) and stomach contents (rumen), scientists have found markers that can help identify the phenotypes of animals that emit less methane and can more easily make use of the nutrients in their diet to gain weight.
The research team comprised veterinarians, biologists, bioinformaticians, epidemiologists and computer scientists. The experiment was conducted with the aim of selecting animals that emit less methane and more efficiently convert nutrients into body mass.
According to the Climate Observatory (Observatório do Clima), a Brazilian network of civil society organizations, farming ranks second in greenhouse gas emissions in Brazil, accounting for 28% of the total. This is mainly due to the large number of beef cattle, which is steadily rising. It was 281.2 million in 2020, the largest number since 2016, according to IBGE, the national statistics bureau.
“We set out to see if we could reduce the volume of greenhouse gas emissions from cattle based on information about their gut microbiome. To do so, we looked for correlations between microbiome components and residual methane emission. However, the main aim of the project was to use samples of bovine feces to find markers [of the emission profile] in their gut microbiome, and in this we were successful,” said Luciana Correia de Almeida Regitano, last author of an article on the subject published in Frontiers in Genetics.
According to Regitano, a specialist in animal genetics and a researcher with EMBRAPA Livestock Southeast, a unit of the Brazilian Agricultural Research Corporation (EMBRAPA), digestion (especially eructation, or belching) is the main source of methane emission in cattle, accounting for more than 90% of the total.
Previous research has found links between bacteria and methane emission, but mostly using ruminal samples, which are very hard to obtain, even for research purposes. “For breeding purposes and genetic improvement, it would be unviable because sample collection involves invasive practices,” she said. “We were surprised by the results of this study, in which we set out to analyze fecal contents in order to correlate them with methane emissions via belching. We didn’t know if we would be successful.”
The researchers analyzed samples from 52 Nelore bulls divided into two groups of 26. One was given a conventional diet based on corn silage, corn, soybean meal, rumen-protected fat and urea concentrate. The other was fed citrus pulp, corn germ, corn germ oil meal, and peanut shell meal, all of which are normally considered industrial waste. Both groups also received mineral supplements, active dry yeast and antibiotics. All animals were housed in a feedlot. The experiment lasted 105 days.
The researchers collected blood samples for DNA extraction and measured methane emissions using an automated feeder fitted with a hood for breath sampling. “The device draws in air and quantifies the gases whenever an animal put its head inside to feed. The results are sent to a data storage facility,” Regitano explained. “It’s expensive, but EMBRAPA invested in the system because several of its research groups are studying the contribution of livestock raising to climate change.”
In the final stage of the experiment, approximately 10 g of stool per animal was obtained two weeks before slaughter, and 50 ml of ruminal content was collected immediately after slaughter. DNA was extracted from the fecal microbiome and amplicons were sequenced. The analysis revealed a total of 5,693 amplicon sequence variants (ASVs).
“To collect bovine microbiome data, we used a technique that lets you make many copies of a specific region in the bacterial or archaeal genome and sequence them. These amplified and sequenced fragments, which we call ASVs, were our units of analysis,” Regitano said.
A microbiome composition analysis resulted in the identification of 30 bacterial and 15 archaeal ASVs as differentially abundant between the two groups of animals. Known as ANCOM, the method compared the microbiomes of the two groups and identified the ASVs whose abundance differed significantly.
An association analysis showed that bacterial ASVs correlated with phenotype variations in terms of the animals’ residual methane emissions and residual feed intake, suggesting their potential use as intervention targets or biomarkers.
“One of the main challenges in this kind of research is the methodology for finding associations or correlations, as thousands of ASVs and metabolites are involved,” Regitano said. “The question here was which method to use to associate the large quantity of data with phenotypes in only 52 animals.”
Measurement of residual methane emissions takes into account variables such as dry matter consumption and permits standardization when gross emissions vary considerably because animals consume different amounts of feed, for example. Residual feed intake refers to the amount consumed by animals to gain weight over and above the amount required to maintain base metabolism. “It’s a measure of dietary efficiency. Some animals need to consume much more feed than you would predict from their base metabolism in order to gain a little weight, and others need to consume less,” Regitano said.
The researchers found that the group fed a conventional diet had a larger variety of microorganisms in their gut microbiome. “We know previous studies said more efficient animals have microbiomes with less diversity, but for this article, we were unable to arrive at a statistically significant difference between the two groups in terms of feed efficiency,” she said, adding that studies exist in the literature correlating bacterial presence and methane emission levels.
“However, the vast majority of studies that make these associations discuss extremes, comparing a group that emits more methane with one that emits less and identifying the bacteria in the latter group, for example. They find associations but can’t quantify the bacterial contribution to methane emissions. We were able to do that, albeit for a relatively small sample of 52 animals,” she said.
The study was supported by FAPESP via a Thematic Project entitled “The Nelore hologenome: implications for beef quality and feed efficiency”, for which Regitano is principal investigator, and via scholarships awarded in Brazil and abroad to Bruno Gabriel Nascimento Andrade, first author of the article.
Emissions and feed efficiency
The researchers identified two bacterial ASVs associated with high residual methane emissions – one in the ruminal microbiome and another in the stool environment. The former belonged to the genus Solobacterium, while on the latter an ASV belonging to the genus Alistipes was associated with a reduction in residual methane emissions. Alistipes bacteria are frequently identified in the bovine microbiome.
The researchers also identified four bacterial ASVs associated with residual feed intake in the ruminal environment. Three correlated with feed inefficiency and one with feed efficiency. They also identified one ASV associated with feed efficiency and one with feed inefficiency in the stool environment.
“In this case, the aim was to select the most efficient animals in using dietary nutrients and in converting them into muscle, so at the same time as we obtained methane emission markers were obtained feed efficiency markers. Data on both traits can be combined to obtain an animal that emits less methane and gains more weight,” Regitano said.
Breeders require large samples for genetic improvement representing major geographic regions and different situations. “That’s why we’re extending this set of samples,” she said. “We’re currently collecting stool samples from a large number of young bulls set to become sires. Having found indicators of emissions and feed efficiency in stool, we’ll extend sample collection. In July, we collected samples from more than 120 animals, and we plan to collect samples from more than 500 animals kept by the Brazilian Association of Zebu Breeders [ABCZ]. We have a partnership with the association and have collected stool samples from their animals since 2019 to obtain a more representative cross-section of the Nelore cattle population.”
EMBRAPA’s samples and those from other sources will first be analyzed separately. “But once we have a reasonable volume, we’ll analyze them together and then we’ll be able to find out whether any given marker serves for various different situations, for example,” Regitano said.
The article “Stool and ruminal microbiome components associated with methane emission and feed efficiency in Nelore beef cattle” is at: www.frontiersin.org/articles/10.3389/fgene.2022.812828/full.