Animal 17 (2023) 100780

Contents lists available at ScienceDirect

Animal
The international journal of animal biosciences

Review: Diving into the cow hologenome to reduce methane emissions

and increase sustainability
Oscar Gonzalez-Recio a,⇑, Natalia Scrobota a,b, Javier López-Paredes c, Alejandro Saborío-Montero d,e,
Almudena Fernández a, Evangelina López de Maturana b,f,g, Beatriz Villanueva a, Idoia Goiri h,
Raquel Atxaerandio h, Aser García-Rodríguez h
a
Departamento de Mejora Genética Animal, INIA-CSIC, 28040 Madrid, Spain
b
Universidad San Pablo-CEU, CEU Universities, Madrid, Spain
c
Confederación de Asociaciones de Frisona Española (CONAFE), Ctra. de Andalucía km 23600 Valdemoro, 28340 Madrid, Spain
d
Escuela de Zootecnia y Centro de Investigación en Nutrición Animal, Universidad de Costa Rica, 11501 San José, Costa Rica
e
Posgrado Regional en Ciencias Veterinarias Tropicales, Universidad Nacional de Costa Rica, 40104 Heredia, Costa Rica
f
Institute of Applied Molecular Medicine (IMMA), Department of Basic Medical Sciences. Facultad de Medicina. Universidad San Pablo-CEU, CEU Universities, ARADyAL, Madrid, Spain
g
Genetic and Molecular Epidemiology Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
h
NEIKER – Instituto Vasco de Investigación y Desarrollo Agrario, Basque Research and Technology Alliance (BRTA), Campus Agroalimentario de Arkaute s/n, 01192 Arkaute, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Interest on methane emissions from livestock has increased in later years as it is an anthropogenic green-
Received 17 September 2022 house gas with an important warming potential. The rumen microbiota has a large influence on the pro-
Revised 6 March 2023 duction of enteric methane. Animals harbour a second genome consisting of microbes, collectively
Accepted 9 March 2023
referred to as the ‘‘microbiome”. The rumen microbial community plays an important role in feed diges-
Available online 17 March 2023
tion, feed efficiency, methane emission and health status. This review recaps the current knowledge on
the genetic control that the cow exerts on the rumen microbiota composition. Heritability estimates
Keywords:
for the rumen microbiota composition range between 0.05 and 0.40 in the literature, depending on the
Feed efficiency
Genetics
taxonomical group or microbial gene function. Variables depicting microbial diversity or aggregating
Genome-Wide Association Study microbial information are also heritable within the same range. This study includes a genome-wide asso-
Holobiont ciation analysis on the microbiota composition, considering the relative abundance of some microbial
Microbiome taxa previously associated to enteric methane in dairy cattle (Archaea, Dialister, Entodinium, Eukaryota,
Lentisphaerae, Methanobrevibacter, Neocallimastix, Prevotella and Stentor). Host genomic regions associated
with the relative abundance of these microbial taxa were identified after Benjamini-Hoschberg correction
(Padj < 0.05). An in-silico functional analysis using FUMA and DAVID online tools revealed that these gene
sets were enriched in tissues like brain cortex, brain amigdala, pituitary, salivary glands and other parts of
the digestive system, and are related to appetite, satiety and digestion. These results allow us to have
greater knowledge about the composition and function of the rumen microbiome in cattle. The state-
of-the art strategies to include methane traits in the selection indices in dairy cattle populations is
reviewed. Several strategies to include methane traits in the selection indices have been studied world-
wide, using bioeconomical models or economic functions under theoretical frameworks. However, their
incorporation in the breeding programmes is still scarce. Some potential strategies to include methane
traits in the selection indices of dairy cattle population are presented. Future selection indices will need
to increase the weight of traits related to methane emissions and sustainability. This review will serve as
a compendium of the current state of the art in genetic strategies to reduce methane emissions in dairy
cattle.
Ó 2023 The Author(s). Published by Elsevier B.V. on behalf of The Animal Consortium. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Implications

This review gathers current knowledge on the genetic control

that the cow exert on the rumen microbiota composition. This is
⇑ Corresponding author. important because rumen microbes are the main drivers of feed
E-mail address: gonzalez.oscar@inia.csic.es (O. Gonzalez-Recio).

https://doi.org/10.1016/j.animal.2023.100780
1751-7311/Ó 2023 The Author(s). Published by Elsevier B.V. on behalf of The Animal Consortium.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
O. Gonzalez-Recio, N. Scrobota, J. López-Paredes et al.
digestion and enteric methane production in ruminants. Methane
emissions should be included in future selection indices in rumi-
nants and in particular, dairy cattle. The microbiota composition
can also be a proxy for methane emission to be included in the
selection indices in populations with a good reference population
for genomic selection.
Introduction
The recent Global Methane Pledge aims to limit methane emis-
sions by 30% in 10 years compared with 2020 levels. Ruminants
play an important role in methane emissions from Agriculture
and Livestock. They have the unique capacity of digesting cellulose
and extract usable energy from it. This process can be viewed as an
anaerobic trophic pyramid in which microbes at each level use the
metabolic residuals from the former as nutrient source. Primary
fermenters decompose complex fibres and generate nutrients
usable by the host and by other secondary fermenters, which in
turn produce methane precursors. During this process, methano-
genic archaea utilise some of these products in a metabolic process
known as methanogenesis which generates methane as a residual
in a process (Crable et al., 2011; Mizrahi et al., 2021). The genome
of this community of microbes (microbiota) is collectively referred
to as the rumen metagenome (Ryu and Davenport, 2022). The
functional and taxonomical composition of the rumen microbiota
of Bos taurus is an important factor contributing to animal perfor-
mance, health, feed efficiency and methane emission (Difford et al.,
2018; Mizrahi and Jami, 2018).
Decreasing enteric methane from ruminants without altering
animal production is desirable both as a strategy to decarbonise
the livestock economy, minimise energy losses and increase effi-
ciency. Variation in microbiome composition, both between and
within species, is due to several factors such as diet, environment
and host genotype (Benson et al., 2010; Spor et al., 2011; Sasson
et al., 2017). Recently, a comprehensive compositional analysis of
the rumen metagenome provided new insights into the association
between microbiota and methane emissions, demonstrating a phe-
notypic association between microbiome composition and
methane production, which is mainly due to the relative abun-
dance (RA) of ciliates and fungi and their effect on the rumen phys-
iological conditions (Saborío-Montero et al., 2020; Lopez-Garcia
et al., 2022). Methane emission is a heritable trait, with heritability
estimates ranging between 0.08 and 0.30, and it is also genetically
correlated to other important traits such as feed efficiency and
milk yield (Breider et al., 2018; López-Paredes et al., 2020). The
microbiota composition has also shown moderate heritabilities
(Difford et al., 2018; Saborío-Montero et al., 2020; 2021). Thus,
methane and microbiota-related traits can be selected in breeding
programmes to reduce methane emissions from ruminants in
future (Lopez-Paredes et al., 2021). Some dairy cattle breeding pro-
grammes have already included novel traits such as feed efficiency
in their breeding goals, but methane traits are still not part of them
(e.g. Byrne et al., 2016). Most selection indices do not specifically
include methane traits, although usually traits such as production
or live weight are included, and they may lead to an (indirect)
effect on methane emissions.
The objective of this review is to describe the current under-
standing on the genetic control exerted by the cow on methane
production as well as on the microbiome, which is responsible
for methanogenesis, and act together with the cow as a hologen-
ome. A genome-wide association study was included to comple-
ment the current state of the art about the genetic architecture
associated to the relative abundance of microorganisms related
to methane production. Then, examples of the inclusion of
methane-related traits in the breeding goals are also presented
2
Animal 17 (2023) 100780
here, and a strategy based on economic weights to develop a selec-
tion index including feed efficiency and methane traits is
suggested.
The cow and its microbiome as a hologenome controlling
methane production
Numerous extrinsic factors influence the composition of the
microbiome, but the role of host genetics has only recently
emerged. The microbiome is important not only for its relationship
with methane, but because it is also associated with other impor-
tant traits such as weight gain, feed efficiency, milk production,
overall animal health and pathogen resistance (Guan et al., 2008;
O’Hara et al., 2020; Xue et al., 2020). Host genetics determines
intrinsic factors that influence the composition of its microbiota.
Consequently, the identification of host genes and their association
with the growth of rumen microbes offers an opportunity in the
study of physiological mechanisms important for host-
microbiome interactions. Several studies in dairy and beef cattle
demonstrated the role of genetics in determining the composition
of the microbiome (Roehe et al., 2016; Difford et al., 2018; Wallace
et al., 2019; Li et al., 2019a; Zhang et al., 2020; Fan et al., 2021;
Saborío-Montero et al., 2021). For example, Roehe et al. (2016)
reported differences in microbial composition between progeny
groups, maintaining the same environmental and dietary condi-
tions. Gonzalez-Recio et al. (2018) compared two cattle breeds
under the same conditions and determined that host genetics
can partially regulate microbiome composition. Difford et al.
(2018) estimated the heritability for more than 3 000 rumen Oper-
ational Taxonomical Units and found that 6% of bacterial and 12%
of archaeal Operational Taxonomic Units had heritabilities signifi-
cantly higher than zero, ranging from 0.16 to 0.44. Wallace et al.
(2019) showed heritabilities ranging between 0.20 and 0.60 for a
group of core microbial taxa. Abbas et al. (2020) demonstrated that
the RA of the most abundant taxa within the rumen microbiome is
a polygenic trait under host genetic control, and identified Opera-
tional Taxonomic Units with relevant heritability (averaging 0.16).
Some of these microbial groups are known to be associated with
methane emissions, for instance, archaea are the main methanogen
group of microbes in the rumen, and live in symbiosis with fungi
and protozoa which provide them substrates and protection from
predator bacteria (Janssen and Kirs, 2008; Saborío-Montero et al.,
2022).
The microbial diversity in the rumen is also heritable. For
instance, Li et al. (2019a) reported heritability for different diver-
sity indices between 0.04 and 0.23, and 0.15 for the ratio between
Firmicutes and Bacteroidetes. However, there is little consensus
about the relationship of microbial diversity with enteric methane
emissions. Variables that aggregate the microbial composition in
principal components or partial least squared components also
showed moderate heritability estimates. Li et al. (2019a) obtained
estimates between 0.15 and 0.25 using Principal Coordinate Anal-
ysis (PCoA). Zhang et al. (2020) found that the proportion of vari-
ance explained by the host genetics for the microbiota variation
using the bacterial (archaeal) first PCoA was 0.22 (0.28)
(SE = 0.10). Fan et al. (2021) showed high heritabilities (0.37 –
0.52) for some core bacterial taxa at the genus level in preweaning,
postweaning, and fattening Angus-Brahman calves. Another study
showed that the variability of the whole metagenome could be
aggregated in several principal components (Saborío-Montero
et al., 2021). The heritability estimates for these principal compo-
nents were consistent across taxonomy level, ranging between
0.30 for the first principal components to 0.11 for the fifth principal
components at phylum level. The complex functionality of the
microbiome can also be reduced in several aggregated variables
O. Gonzalez-Recio, N. Scrobota, J. López-Paredes et al.
that are also heritable. Saborío-Montero et al., (2021) showed a
heritability estimate of 0.34 for the first principal component
aggregating the functional information from Kyoto Encyclopedia
of Genes and Genomes (KEGG) functional classification. Interest-
ingly, these aggregated variables showed strong genetic correla-
tions (>0.70) with methane emissions.
These previous results evidence that cow genes and their vari-
ants determine some conformation and physiological characteris-
tics in the animal which favours (or impair) the proliferation of
certain microorganisms. Thus, efforts have been made to detect
regions in the cow genome that may regulate the growth of certain
microbes in the rumen and therefore be associated with the com-
position of the microbiome. Fan et al., (2021) reported significant
Single Nucleotide Polymorphisms (SNPs) associated with the
microbiome composition. These SNPs were located in genes
involved in metabolism, immunity or gastrointestinal tract devel-
opment. They also found associations with the genes ZNF641,
PFKM, NSMAF, CTNNAL1 and EPB41L4B. In a similar study, Li et al.
(2019b) also found TBC1D5, SMOC1 and RAPH1 associated with
Ruminococcus, OPTN, MCM10, UCMA with Spirochaetes; and a non-
coding region in BTA19 associated with Lentisphaerae, among other
non-coding regions associated with the RA of several microbes in
the rumen. Most of these genes are related to different types of
metabolism and cellular development.
Genetic architecture of microbial composition, in the sense of
the complex relationship between cow genes and microbes rela-
tive abundance as a phenotypic trait, is not yet well understood,
and more research is needed. In the following section, we intend
to provide additional evidence of how the cow genome may be
controlling the microbiota composition.
Genome-wide association study for the microbiota composition
Study population
Data used for this work came from a cohort of 439 Holstein
cows sampled from 14 different herds in Northern Spain between
May 2018 and June 2019. The data set is described in Saborío-
Montero et al. (2021) and Lopez-Garcia et al. (2022). In short,
rumen content samples were extracted through oroesophageal
tube and immediately frozen using nitrogen liquid vapours and
storage at
80 °C at the Animal Breeding Department at INIA-
CSIC (Madrid, Spain) until DNA was extracted using the DNeasy
Qiagen PowerSoil kit and sequenced using Oxford Nanopore Tech-
nologies. Quality control was applied to remove sequences with
Phred quality score (Q score) < 7 and length < 150 bp. Sequence
analysis was performed using the SqueezeMeta pipeline for long
reads (Tamames and Puente-Sánchez, 2019).
All cows were genotyped using the Illumina 50 k SNP BeadChip.
Genotypes were filtered out by minor allele frequency < 0.05, and
call rate < 95%. Missing genotypes were imputed using Beagle 5.4
by the Spanish Holstein Association CONAFE (Jiménez-Montero
et al., 2012).
Genotypes were imputed to whole genome sequencing using
the 1 000 bull genome reference population (Hayes and
Daetwyler, 2019). After filtering by minor allele frequency > 0.01
and quality control, 13 157 778 SNPs remained for genome-wide
association analyses.
The following genera were kept for subsequent Genome-Wide
Association Analysis (GWAS) because they have been previously
reported to be associated to methane production (Saborío-
Montero et al., 2020 and 2021; Lopez-Garcia et al., 2022): Stentor,
Neocallimastix, Dialister, Prevotella, Methanobrevibacter, Entodinium
and Lentisphaerae. The relative abundances of Archaea and Eukary-
ote were also analysed.
3
Animal 17 (2023) 100780
Statistical model
The statistical model used for each GWAS was as follows:
y ¼ 10 l þ bx þ g þ e
where y was the phenotype (relative abundance of the preadjusted
by other environmental effects), 1 is a vector of ones of correspond-
ing dimension, l is the intercept, b is the additive effect of the can-
didate SNP to be tested for association, x is the SNP genotype
dosage, g is the polygenic effect assumed to be distributed as
g  N(0,Gr2g ), where G is the genomic relationship matrix
(VanRaden, 2008) and r2g is the genomic variance, and e is the vec-
tor of residuals.
The GWAS was implemented using the Genome-wide Complex
Trait Analysis software (version 1.93.2) (Yang et al., 2011). All anal-
yses were performed at the CESGA high computing performance
centre (Galicia, Spain).
Significant SNPs of each genus and superkingdom were anno-
tated in genes. The annotation of significant SNPs was determined
with Ensembl’s biomaRt package for R (Durinck et al., 2009), using
the ARS-UCD1.2 reference genome, accessed through Ensembl
(https://www.ncbi.nlm.nih.gov/assembly/GCF_002263795.1/), and
the Ensembl Genes 106 database (Cunningham et al., 2022). A total
of 50 000 bases upstream and downstream of significant SNPs
were considered for gene annotation. SNPs with an adjusted P-
value <0.05 were considered significant. The P-value was adjusted
by the Bonferroni method or false discovery rate (FDR) to correct
for multiple comparisons (Benjamini and Hochberg, 1995).
PubMed and The National Center for Biotechnology Information
database were used to investigate the biological function of the sig-
nificant genes related to the rumen microbiota. The online tools
FUMA v1.3.8 (Watanabe et al., 2017), https://fuma.ctglab.nl) and
DAVID 6.8 (Huang et al., 2009), https://david.ncifcrf.gov/home.
jsp) were utilised for enrichment analysis and functional annota-
tion of genes. For FUMA, the GENE2FUNC tool was used to perform
gene set enrichment analyses using among other database expres-
sion data from the Genotype-Tissue Expression (GTEx) project,
which comes from 54 non-diseased tissue samples from nearly
1000 human individuals, and gene sets included in the molecular
signatures database (Subramanian et al., 2005; Liberzon et al.,
2011). DAVID, on the other hand, employs the ARS-UCD1.2 B. tau-
rus genome database (https://www.ncbi.nlm.nih.gov/assembly/
GCF_002263795.1/). In addition, the Uniprot database (Bateman
et al., 2021) is consulted to obtain functional information on the
proteins encoded by the genes of interest, and KEGG to determine
the metabolic pathways involved.
Significant single nucleotide polymorphisms and gene identification
Fig. 1 shows the Manhattan plots representing the -log10 P-
values of the entire GWAS on a genome-wide scale according to
their genome position using the qqman R package (Turner,
2018). After a stringent significance threshold, we found significant
SNPs associated to the relative abundance of Dialister (41), Len-
tisphaerae (627) and Neocallimastix (55). The largest number of
genes with significant SNPs was found for Lentisphaerae (78 genes).
Three genes were associated to Neocallimastix (RNF186, TMCO4,
PAX7) and 2 with Dialister (FBRSL1, PRP2). Although the Manhattan
plots showed several peaks for Stentor, Methanobrevibacter, and
Entodinium, they were not significant at the threshold level
required in this study. Larger data sets might increase the statisti-
cal power in future studies.
O. Gonzalez-Recio, N. Scrobota, J. López-Paredes et al. Animal 17 (2023) 100780

Fig. 1. Manhattan plots for the relative abundance of Archaea, Eukaryote, Dialister, Lentisphaerae, Methanobrevibacter, Neocallimastix and Stentor in 439 Spanish Holstein cows.
Relative abundance was preadjusted by contemporary group of herd-pen, lactation number and stage of lactation. Significance level after False Discovery Rate (5%) and
Bonferroni correction are depicted as blue and red horizontal lines, respectively. Abbreviation: SNP = Single Nucleotide Polymorphism.

Functional in-silico analyses
We performed a functional in-silico analysis with the eighty-
three genes harbouring the genetic variants associated with the
RA of any of the taxa analysed using two bioinformatics tools:
DAVID and FUMA.
Fig. 2 shows the gene expression heatmap of the 83 genes asso-
ciated with RA of Dialister, Letisphaerae and Neocallimastix in the 54
tissue types of GTEx project v8. The angiogenesis hallmark gene
set, i.e. these genes show well-defined biological processes and
consistent expression for angiogenesis processes, was significantly
enriched (Adjusted P-value < 0.05) with three genes (ITGAV,
COL3A1 and COL5A2). In addition, COL3A1 and COL5A2, along with
COL8A1, were significant for gene ontology of cellular components
(GO-CC), specifically for collagen trimer complex formation (as
they are involved in protein digestion and absorption). Genes asso-
ciated with immunological signatures were also found: ITGAV,
ARL6, CRYBG3, CRYBG3, CRYBG3, CLDND1, GPR15, ALCAM and BTLA
(Watanabe et al., 2017). Interestingly, TFF1 (Trefoil Factor 1) and
TFF2 (Trefoil Factor 12) were only expressed in the stomach from
the FUMA analysis based on humans. Both genes are involved in
the maintenance of gastrointestinal epithelium GO biological process.
In B. Taurus, the COL3A1, COL5A2 and COL8A1 genes are involved in
protein digestion and absorption (Sherman et al., 2022). Acid pH
alters the composition of the ruminal microbiota, increasing the
availability of fermentable substrates and enhancing the growth
of proteobacteria related to propionate fermentation and starch
degradation (Xu et al., 2021). Excessive fermentation of
carbohydrate-rich diets may cause ruminal acidosis, which
increases lactic acid production and thus lowers ruminal pH. An
acidotic rumen is also characterised by a decrease in ciliated proto-
zoa (Nagaraja and Titgemeyer, 2007), i.e., the relative abundance of
eukaryotes decreases. Saliva is an important buffer for the rumen,
and expression in the salivary glands may be an important factor
regulating rumen pH.
Intriguingly, using the human tissue expression data from GTEx
project (v8), we found that the differentially expressed gene (DEG)
sets in the pituitary and stomach tissues from humans were signif-
icantly enriched with our gene set (Figs. 2 and 3). These tissues are
markedly related to appetite, satiety and digestion. Considering
only the up-regulated DEG gene sets, our gene set were enriched
in five tissues (brain cortex, brain nucleus accumbens basal gan-
glia, brain putamen basal ganglia, brain amigdala and brain cau-
date basal ganglia). Brain is also an important organ determining
satiety, voracity, and has been extensively linked to the gut micro-
biota (Carabotti et al., 2015). Considering only the down-regulated
DEG genes, the set expressed in artery tibial was significantly
enriched (Fig. 3).

4
O. Gonzalez-Recio, N. Scrobota, J. López-Paredes et al. Animal 17 (2023) 100780

Fig. 2. Heatmap showing the log2 transformed expression value of the 83 genes harbouring genetic variants significantly associated with the relative abundance of Dialister,
Letisphaerae and Neocallimastix considering 54 tissue types of GTEx v8 project. These 83 genes were obtained from the significant SNPs associated to the relative abundance
of these microbes in a genome-wide association study using 439 Spanish Holstein cows. Abbreviation: SNP = Single Nucleotide Polymorphism.

DAVID found three clusters based on redundant annotation
terms at FDR < 0.05 (see Table 1). We highlight the first one includ-
ing three GO molecular function terms RNA polymerase II core pro-
moter proximal region sequence-specific DNA binding (ZNF175,
ZNF350, ZNF581, ZNF580, ZNF628, ZNF280A, ZNF280B, ZNF524,
ZNF521, ZNF784), transcriptional activator activity (ZNF175,
ZNF580, ZNF628, ZNF521, ZNF784), and RNA polymerase II tran-
scription regulatory region sequence-specific binding and metal
ion binding (RNFT2, ZNF350, ABLIM3, ZNF280A, ZNF280B, LENG9,
DTX1, RNF186, ZNF784). The second cluster includes the Natural
killer cell-mediated cytotoxicity KEGG pathway, which includes
four (KIR3DS1, IGLON5, KIR3DL2, FCAR) genes from our gene set.
These results suggest that the RA of microbes in the rumen is
genetically regulated as a polygenic/infinitesimal trait, with many
5
genes acting with a small effect. Few genomic regions are associ-
ated with the RA of these microbes determining feed efficiency
and methane production in the rumen, although they explained a
small proportion (<1%) of the genetic variance. The genes con-
tained in these regions are overexpressed in the brain-gut system,
the rumen, pituitary, and salivary glands, and they are associated
to appetite and rumen environmental conditions. Larger data sets
might reveal new significant regions or increase the amount of
genetic variance explained.
Our results show additional evidence of candidate genomic
regions controlling the microbiota composition. We recognise that
the limited sample size may prevent the detection of small effects.
Nonetheless, given the difficulties to obtain a large data set that
provides enough statistical power to genome-wide association
O. Gonzalez-Recio, N. Scrobota, J. López-Paredes et al. Animal 17 (2023) 100780

Fig. 3. Barplot relating the –log10 p-value resulting from the enrichment analysis of the 83 genes harbouring genetic variants significantly associated with the relative
abundance of Dialister, Letisphaerae and Neocallimastix with the sets of differentially expressed genes (DEGs) in each of the 54 tissue types of GTEx project v8. These 83 genes
were obtained from the significant SNPs associated to the relative abundance of these microbes in a genome-wide association study using 439 Spanish Holstein cows.
Abbreviation: SNP = Single Nucleotide Polymorphism.

Table 1
Clusters of biological functions for the significant single nucleotide polymorphism of each taxa according to the Bos taurus genome obtained with The Database for Annotation,
Visualisation and Integrated Discovery (Huang et al., 2009).

Cluster Genes Fold Enrichment False Discovery Rate

IPR013087:Zinc finger C2H2-type/integrase ZNF175, ZNF350, ZNF581, ZNF580, ZNF628, ZNF280A, ZNF280B, 5.35 0.001
DNA-binding domain ZNF579, FIZ1, ZNF524, ZNF521, ZNF784
IPR013151:Immunoglobulin KIR3DS1, IGLON5, KIR3DL2, FCAR 18.80 0.001
KW-1133  Transmembrane helix CLDND2, KIR3DS1, TTYH1, TMPRSS3, SPRING1, NAT14, VSIG10L, CACNG6, 1.62 0.049
CACNG8, HAS1, VSTM1, SYT5, LIM2, TMEM150B, TMEM86B, SIGLEC10,
TMEM190, NKG7, KIR3DL2, TMCO4, NCR1, B3GNT4, RNF186, SPACA6, RPL28

studies, we believe our results significantly contribute to the state
of the art on the hologenome topic, and constitute a starting point
to future efforts and international collaborations to obtain larger
data sets. The available evidence of the host genetic control on
methane emissions and the rumen microbiome compositions
strongly suggests that these traits may be incorporated into breed-
ing programmes to modulate the rumen microbial composition in
future generations for a more efficient feed digestion and lower
methane production.
Inclusion of methane-related traits in the selection indices
Selection indexes are used in breeding programmes to obtain
some genetic gain on the traits included in the breeding goal.
The inclusion of methane-related traits into the breeding goals in
dairy cattle has been of increasing interest in recent years. The
6
main reasons are its social and environmental importance, and
its potential association with feed efficiency. Nonetheless, no prac-
tical implementation is broadly applied yet due to several reasons.
For example: scarce availability of methane data, lack of standards
in the measurement protocols, unclear definition of the most suit-
able trait of interest and difficulties for calculating an economic
value for methane traits. The correlated genetic response obtained
through proxies and the impact of genomic selection need also to
be evaluated (Brito et al., 2020). In particular, methane emissions
show an antagonistic correlation with fat yield, with a genetic cor-
relation estimated between 0.20 and 0.30 (Dijkstra et al., 2011;
Pszczola et al., 2019; López-Paredes et al., 2020).
Different strategies have been proposed to include methane
emissions in the breeding goal in dairy cattle. For instance,
González-Recio et al. (2020) proposed several strategies for includ-
ing methane mitigation into the breeding goal and reducing the
energy loss in dairy cattle. They concluded that methane should
O. Gonzalez-Recio, N. Scrobota, J. López-Paredes et al.
be incorporated into the breeding goal with a relative weight larger
than 25% to achieve the mitigation targets agreed at the 2021
United Nations Climate Change Conference (COP26) if no other
strategies than genetics are considered. In addition, Manzanilla-
Pech et al. (2021) proposed several indices in an international col-
laboration which included data from Australia, Denmark, Canada,
and Switzerland from Holstein population. They combined differ-
ent direct and residual feed efficiency traits, BW and production
under different scenarios according to the economic values of
methane traits (from 0 to
0.60€). They concluded that including
residual feed intake with a negative economic value in the breed-
ing goal reduces the expected response of methane production
compared with the base scenario (only including and economic
value for energy-corrected milk of 1.00€), meaning that selecting
more feed-efficient animals is also selecting for lower methane
emitting animal. Save feed and methane emission were also pro-
posed as additional traits in the current selection indexes for the
Australian and Dutch national breeding goal, which includes 15
traits from milk production, milk components, feed efficiency,
health, reproduction, longevity, calving, and conformation achiev-
ing a reduction of 24% of methane emissions in 2050 (Byrne
et al., 2016; de Haas et al., 2021). A total of 12 possible selection
indexes were recently considered in the Australian dairy popula-
tion by (Richardson et al., 2022). This study also showed that a
40–50% reduction on methane emissions is possible through selec-
tive breeding at the expense of a relatively low loss in percentage
of economic gain, but still positive.
Other related traits such as rumination time have been analysed
as proxies (Byskov et al., 2015; Beauchemin, 2018), although not
yet proposed as a trait in any selection index. Further, the role of
core microbiome in the optimisation of the digestion process has
also been incorporated into the paradigm in recent years (Difford
et al., 2018; Saborío-Montero et al., 2020 and 2021; Lopez-
Paredes et al., 2021). These related traits pose interesting alterna-
tives for accelerating this genetic response and to achieve the tar-
get methane mitigation and the reduction of feeding demand for
the upcoming years.
It must be pointed out that the incorporation of these traits in the
breeding goal needs to be carefully considered because it might
imply decelerating the farm profitability and reduced genetic pro-
gress for production traits, given the antagonistic genetic correlation
between methane and some of these traits. However, a balanced
selection is possible for the simultaneous improvement of all traits
in the breeding goal. Previous experiences have demonstrated the
possibility of improving antagonistic traits simultaneously. The most
notable example is possibly female fertility, which has been geneti-
cally improved in later years despite its antagonistic correlation with
milk yield (Ma et al., 2019). The breeding strategies revisited here
show the possibility of a balanced selection including methane traits
in the breeding goals of dairy cattle. However, establishing a balanced
breeding goal while considering all productive systems is cumber-
some, as there is an important interaction between the animal, the
microbiome and the type of ration provided.
Considering all these limitations, selective breeding (either
genetic or genomic) can be applied at a low cost, and its effects
on the population are permanent and cumulative. They can apply
a more balanced selection even for antagonistic traits (e.g.
methane and milk fat). Residual traits (methane phenotype
adjusted by production or intake traits) are appealing selection
candidates because they are not correlated to other traits of inter-
est. This will cause the genetic response on methane to be indepen-
dent of production traits. In conclusion, selection for more
sustainable animals involves great complexity due to economic
and biological factors, but environmental consequences should
be considered in the development of selection indices in the fol-
lowing years under new balanced strategies.
7
Animal 17 (2023) 100780
Future perspectives
The climate emergency requires action from all sectors to
reduce their impact on global warming and environment. Dairy
cattle can contribute to this mitigation by reducing methane emis-
sions. Breeding has a great potential for this contribution within
10–20 years, as methane traits are controlled by the host geno-
types with moderate heritabilities. Part of the variability of
methane is also controlled by the rumen metagenome. We showed
that the metagenome composition is a quantitative trait partially
controlled by the host genotype, with some genomic regions of
interest controlling it. In particular, these genomic regions are
mainly expressed in organs and tissues related to appetite, satiety
and gastrointestinal functionality.
This review shows that the cow hologenome may be modulated
to increase feed efficiency and reduce methanogenesis in the
rumen. There are three main challenges that need to be overcome:
(1) data recording on methane emissions to build a large enough
reference population, (2) determining the more appropriate set of
traits to be included in the selection indices, and (3) a balanced
inclusion of methane-related traits in the selection indices while
limiting undesirable effects on other traits such as fat yield.
It may take some time until these challenges are perfectly tack-
led. However, there is no time to lose to start making progress and
apply actions based on the current knowledge, which can be redi-
rected as more results and evidence are gathered across interna-
tional efforts and collaborations.
Ethics approval
This study was conducted in accordance with Spanish Royal
Decree 53/2013 for the protection of animals used for experimen-
tal and other scientific purposes and was approved by the Basque
Institute for Agricultural Research and Development Ethics Com-
mittee (Neiker-OEBA-2017–004) on 28 March 2017.
Data and model availability statement
The raw data underlying the metagenomic data are available on
the ENA website and can be accessed with bioproject accession No.
PRJNA789746 [https://www.ebi.ac.uk/ena/browser/view/
PRJNA789746. Accessed 5 September 2022.]. Other data were not
deposited in an official repository and can be requested from the
METALGEN project.
Author ORCIDs
Oscar González-Recio: https://orcid.org/0000-0002-9106-
4063.
Natalia Scrobota: https://orcid.org/0009-0005-7492-3347.
Javier López-Paredes: https://orcid.org/0000-0003-1507-2595.
Alejandro Saborío-Montero: https://orcid.org/0000-0002-
9840-0058.
Almudena Fernández: https://orcid.org/0000-0002-4495-
9366.
Evangelina López de Maturana: https://orcid.org/0000-0001-
9425-3911.
Beatriz Villanueva: https://orcid.org/0000-0003-4645-8853.
Idoia Goiri: https://orcid.org/0000-0002-6522-0085.
Raquel Atxaerandio: https://orcid.org/0000-0002-8906-8516.
Aser García-Rodríguez: https://orcid.org/0000-0001-5519-
6766.
O. Gonzalez-Recio, N. Scrobota, J. López-Paredes et al.
Author contributions
A.G.R., R.A., I.G. and O.G.R. designed the sampling. A.G.R., R.A.,
and I.G. obtained the samples. A.S.M and O.G.R. filtered and pre-
pared the phenotypic and metagenomic data. O.G.R. filtered and
prepared the genotype data. A.F. and O.G.R. performed genotype
imputation, O.G.R. implemented the GWAS. N.S. and E.L.M. per-
formed the in-silico analyses. O.G.R. conceived the study and
designed the GWAS. O.G.R., B.V. and A.G.R. contributed to the
acquisition of funding and resources. J.L.P, and O.G.R. drafted the
first version of the manuscript. All authors helped in writing and
configuring the last version of the manuscript.
Declaration of interest
The authors state that they have no competing interests.
Acknowledgements
We thank the regional Holstein Associations and farmers for
collaborating in the project. Computational support from the
High-Performance Computing Centre in Galicia (CESGA, Spain) is
acknowledged. We also thank the 1000 bull genomes consortium
for providing the reference population for whole genome sequence
imputation.
Financial support statement
This research was financed by RTA2015-00022-C03 (METAL-
GEN) project from the National Plan of Research, Development
and Innovation 2013–2020 and the Department of Economic
Development and Competitiveness (Madrid, Spain). BV thanks
funding from MCIN/ AEI /10.13039/501100011033 (Project
PID2020-114426GB-C22).
Transparency Declaration
This article is part of a supplement entitled Selected keynote
lectures of the 73rd Annual Meeting of the European Federation
of Animal Science (Porto, Portugal) supported by the Animal
Consortium.
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