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Simple Summary Metabolic disorders represent a crucial problem in early lactating dairy cows, which lead to major economic losses at the herd level. To allow the prompt detection of metabolic dysfunction with noninvasive and ready-to-use methods on the farm, milk matrix represents the best option. Among milk fine components, the fatty acid profile represents a fingerprint of the cow's nutritional and metabolic status, being a suitable indicator of metabolic imbalance at the cow level. We performed an association study between milk fatty acid profile and a set of metabolic indicators, such as body condition score, ultrasound liver measurements, and blood metabolites, from 297 Holstein-Friesian cows. We extracted a few latent variables able to explain specific biological mechanisms from the milk fatty acid profile. Then, we explored the associations between these new synthetic variables, namely the factors, and the morphometric, ultrasonographic and hematic indicators of immune and metabolic status. The significant associations of fatty acid factors with blood and ultrasound indicators of inflammation and hepatic load showed the capacity of fatty acids to reflect the energy metabolic status of lactating cows, suggesting their potential usefulness as markers of digestive alterations and metabolic variations in cows during the critical period of early lactation. Dairy cows have high incidences of metabolic disturbances, which often lead to disease, having a subsequent significant impact on productivity and reproductive performance. As the milk fatty acid (FA) profile represents a fingerprint of the cow's nutritional and metabolic status, it could be a suitable indicator of metabolic status at the cow level. In this study, we obtained milk FA profile and a set of metabolic indicators (body condition score, ultrasound liver measurements, and 29 hematochemical parameters) from 297 Holstein-Friesian cows. First, we applied a multivariate factor analysis to detect latent str

Indicine and taurine subspecies present distinct morphological traits as a consequence of environmental adaptation and artificial selection. Although the two subspecies have been characterized and compared at genome-wide level and at specific loci, their epigenetic diversity has not yet been explored. In this work, Reduced Representation Bisulphite Sequencing (RRBS) profiling of the taurine Angus (A) and indicine Nellore (N) cattle breeds was applied to identify methylation differences between the two subspecies. Genotyping by sequencing (GBS) of the same animals was performed to detect single nucleotide polymorphisms (SNPs) at cytosines in CpG dinucleotides and remove them from the differential methylation analysis. A total of 660,845 methylated cytosines were identified within the CpG context (CpGs) across the 10 animals sequenced (5 N and 5 A). A total of 25,765 of these were differentially methylated (DMCs). Most DMCs clustered in CpG stretches nearby genes involved in cellular and anatomical structure morphogenesis. Also, sequences flanking DMC were enriched in SNPs compared to all other CpGs, either methylated or unmethylated in the two subspecies. Our data suggest a contribution of epigenetics to the regulation and divergence of anatomical morphogenesis in the two subspecies relevant for cattle evolution and sub-species differentiation and adaptation.

In contrast to nuclear markers routinely used for genomic selection, mitogenome information has been underutilized for breeding and biodiversity management of cattle populations. Our main goal was to promote the efficient use of mitogenome SNPs contained in commercial high-throughput SNP arrays. In collaboration with NEOGEN Genomics (Lincoln, NE, USA), we integrated 310 SNPs into the commercial GGP Bovine 100K SNP array. In doing so, we demonstrated how mitogenome SNPs can be used in high-throughput arrays to (i) analyze population structure and diversity, (ii) classify bovine haplogroups and identify introgression and/or upgrading, (iii) screen and identify pedigree defects, (iv) impute mitogenome information on maternal lineages to increase statistical power in estimating the effects of mitogenome variation on quantitative production traits, and (v) identify deleterious mutations found in humans. In addition, we have developed protocols and pipelines inte-grated with the Magellan v2.0 software to enable efficient and routine use of mitogenome information in cattle breeding and genetic diversity management. Finally, we have highlighted some other interesting opportunities for the use of mitogenome information in the near future.

Better understanding of the molecular mechanisms behind bovine mastitis is fundamental for improving the management of this disease, which continues to be of major concern for the dairy industry, especially in its subclinical form. Disease severity and progression depend on numerous aspects, such as livestock genetics, and the interaction between the causative agent, the host, and the environment. In this context, epigenetic mechanisms have proven to have a role in controlling the response of the animal to inflammation. Therefore, in this study we aimed to explore genome wide DNA methylation of milk somatic cells (SC) in healthy cows (n = 15) and cows affected by naturally occurring subclinical mastitis by Streptococcus (Strep.) agalactiae (n = 12) and Prototheca spp. (n = 11), to better understand the role of somatic cells methylome in the host response to disease. Differentially methylated regions (DMR) were evaluated comparing: i) Strep. agalactiae-infected vs. healthy; ii) Prototheca-infected vs. healthy, and iii) mastitis vs. healthy and iv) Strep. agalactiae-infected vs. Prototheca-infected. The functional analysis was performed at 2 levels. To begin with, we extracted differentially methylated genes (DMG) from promoter - DMR, which were analyzed using the Cytoscape ClueGO plug-in. Coupled with this DMG-driven approach, all the genes associated with promoter - methylated regions were fed to the Pathifier algorithm. From the DMR analysis, we identified 1,081 hypermethylated and 361 hypomethylated promoter regions in Strep. agalactiae infected animals, while 1,514 hypermethylated and 358 hypomethylated promoter regions were identified in Prototheca infected animals, when compared with the healthy controls. When considering infected animals as a whole group (regardless of the pathogen), we found 1,576 hypermethylated and 460 hypomethylated promoter regions. Both pathogens were associated with methylation differences in genes involved in pathways related to meio

Background: Subclinical intramammary infection (IMI) represents a significant problem in maintaining dairy cows’ health. Disease severity and extent depend on the interaction between the causative agent, environment, and host. To investigate the molecular mechanisms behind the host immune response, we used RNA-Seq for the milk somatic cells (SC) transcriptome profiling in healthy cows (n = 9), and cows naturally affected by subclinical IMI from Prototheca spp. (n = 11) and Streptococcus agalactiae (S. agalactiae; n = 11). Data Integration Analysis for Biomarker discovery using Latent Components (DIABLO) was used to integrate transcriptomic data and host phenotypic traits related to milk composition, SC composition, and udder health to identify hub variables for subclinical IMI detection. Results: A total of 1,682 and 2,427 differentially expressed genes (DEGs) were identified when comparing Prototheca spp. and S. agalactiae to healthy animals, respectively. Pathogen-specific pathway analyses evidenced that Prototheca’s infection upregulated antigen processing and lymphocyte proliferation pathways while S. agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle, and carbohydrate and lipid metabolism. The integrative analysis of commonly shared DEGs between the two pathogens (n = 681) referred to the core-mastitis response genes, and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells (r 2 = 0.72), followed by the udder health (r 2 = 0.64) and milk quality parameters (r 2 = 0.64). Variables with r ≥ 0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cytohubba plug-in. The genes in common between DIABLO and cytohubba (n = 10) were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals (sensitivity > 0.89, specificity > 0.81, accuracy

Background: During epididymal transit spermatozoa acquire specific morphological features which enhance their ability to swim in a progressive manner and interact with the oocytes. At the same time, sperm cells undergo specific molecular rearrangements essential for the fertilizing sperm to drive a correct embryo development. To assess epigenetic sperm changes during epididymal maturation, the caput, corpus and cauda epididymis sperm tracts were isolated from eight bulls and characterized for different sperm quality parameters and for CpG DNA methylation using Reduced Representation Bisulfite Sequencing (RRBS) able to identify differentially methylated regions (DMRs) in higher CpG density regions. Results: Caput sperm showed significant variation in motility and sperm kinetics variables, whereas spermatozoa collected from the corpus presented morphology variation and significant alterations in variables related to acrosome integrity. A total of 57,583 methylated regions were identified across the eight bulls, showing a significantly diverse distribution for sperm collected in the three epididymal regions. Differential methylation was observed between caput vs corpus (n = 11,434), corpus vs cauda (n = 12,372) and caput vs cauda (n = 2790). During epididymal transit a high proportion of the epigenome was remodeled, showing several regions in which methylation decreases from caput to corpus and increases from corpus to cauda. Conclusions: Specific CpG DNA methylation changes in sperm isolated from the caput, corpus, and cauda epididymis tracts are likely to refine the sperm epigenome during sperm maturation, potentially impacting sperm fertilization ability and spatial organization of the genome during early embryo development.

Background: Blood metabolic profiles can be used to assess metabolic disorders and to evaluate the health status of dairy cows. Given that these analyses are time-consuming, expensive, and stressful for the cows, there has been increased interest in Fourier transform infrared (FTIR) spectroscopy of milk samples as a rapid, cost-effective alternative for predicting metabolic disturbances. The integration of FTIR data with other layers of information such as genomic and on-farm data (days in milk (DIM) and parity) has been proposed to further enhance the predictive ability of statistical methods. Here, we developed a phenotype prediction approach for a panel of blood metabolites based on a combination of milk FTIR data, on-farm data, and genomic information recorded on 1150 Holstein cows, using BayesB and gradient boosting machine (GBM) models, with tenfold, batch-out and herd-out cross-validation (CV) scenarios. Results: The predictive ability of these approaches was measured by the coefficient of determination (R2). The results show that, compared to the model that includes only FTIR data, integration of both on-farm (DIM and parity) and genomic information with FTIR data improves the R2 for blood metabolites across the three CV scenarios, especially with the herd-out CV: R2 values ranged from 5.9 to 17.8% for BayesB, from 8.2 to 16.9% for GBM with the tenfold random CV, from 3.8 to 13.5% for BayesB and from 8.6 to 17.5% for GBM with the batch-out CV, and from 8.4 to 23.0% for BayesB and from 8.1 to 23.8% for GBM with the herd-out CV. Overall, with the model that includes the three sources of data, GBM was more accurate than BayesB with accuracies across the CV scenarios increasing by 7.1% for energy-related metabolites, 10.7% for liver function/hepatic damage, 9.6% for oxidative stress, 6.1% for inflammation/innate immunity, and 11.4% for mineral indicators. Conclusions: Our results show that, compared to using only milk FTIR data, a model integrating milk FTIR spe

Milk fatty acid (FA) profile is a clear example of complex and multiple correlated traits whose genetic basis is difficult to assess. Although genome-wide association (GWA) studies have been successful in the identification of significant genetic variants for complex traits, when correlated phenotypes are analysed separately, the outcomes are difficult to compare and interpret in a metabolic context. Here, we performed a multivariate factor analysis (MFA) on Italian Simmental and Italian Holstein milk fat profiles to extract latent unobserved factors able to explain correlation structure and common metabolic function among different FAs. Individual factor scores obtained by MFA were used to perform a single-SNP based GWA. In both breeds, MFA was able to extract ten latent factors with specific biological meaning, notably: de novo synthesis, desaturation activity and biohydrogenation. The GWA result confirmed the increased power of joint association analysis on multiple correlated traits and allowed us to identify major candidate genes with well-documented function consistent with the metabolic classification of factors obtained, such as DGAT1, FASN and SCD. © 2020, Institute of Plant Genetics, Polish Academy of Sciences, Poznan.

The genotype of an organism is stable throughout its life; however, its epigenome is dynamic and can be altered in response to environmental factors, such as diet. Inheritance of acquired epigenetic modifications by the next generation occurs through the germline, although the precise mechanisms remain to be elucidated. Here, we used a sheep model to evaluate if modification of the maternal diet (CTR; control, UND: undernutrition; FA: undernutrition and folic acid supplementation) during the peri-conceptional period affects the genome-wide methylation status of the gametes of male offspring. Sperm DNA methylation, measured by Reduced Representation Bisulfite Sequencing (RRBS), identified Differentially Methylated Regions (DMR) in offspring that experienced in utero undernutrition, both in UND (244) and FA (240), compared with CTR. Gene ontology (GO) analysis identified DMRs in categories related to sperm function, therefore we investigated whether the fertilizing capacity of the semen from the three groups differed in an in vitro fertilization assay. Spermatozoa from the undernourished groups showed lower motility and sperm chromatin structure abnormalities, represented by a higher percentage of DNA fragmentation and an increased number of immature cells, compared with CTR. While good quality blastocysts were obtained from all three groups, the proportion of embryos reaching the blastocyst stage was reduced in the UND vs CTR, an effect partially rescued by the FA treatment. The data reported here show that nutritional stress during early pregnancy leads to epigenetic modifications in the semen of the resulting offspring, the effects of which in next generation remain to be elucidated. © 2020 Society for Reproduction and Fertility.

Genomic selection (GS) reports on milk fatty acid (FA) profiles have been published quite recently and are still few despite this trait represents the most important aspect of milk nutritional and sensory quality. Reasons for this can be found in the high costs of phenotype recording but also in issues related to its nature of complex trait constituted by multiple genetically correlated variables with low heritabilities. One possible strategy to deal with such constraint is represented by the use of dimension reduction methods. We analysed 40 individual FAs from Italian Brown Swiss, Holstein and Simmental milk through multivariate factor analysis (MFA) to study the genetics of milk FA-related latent variables (factors) and assess their potential use in breeding. A total of nine factors were obtained, and their genetic parameters were inferred under a Bayesian framework using two statistical approaches: the classical pedigree best linear unbiased prediction (ABLUP) and the single-step genomic BLUP (ssGBLUP). The resulting factorial solutions were able to represent groups of FAs with common origin and function and can be considered concise pathway-level phenotypes. The heritability (h2 ) values showed relevant variations across different factors in each breed (0.03 ≤ h2 ≤ 0.38). The accuracies of breeding values predicted were low to high, ranging from 0.13 to 0.72 and from 0.18 to 0.74 considering the pedigree and the genomic model, respectively. The gain in accuracy in genetic prediction due to the addition of genomic information was ~30% and ~5% in validation and training groups respectively, confirming the contribution of genomic information in yielding more accurate predictions compared to the traditional ABLUP methodology. Our results suggest that MFA in combination with GS can be a valuable tool in dairy cattle breeding and deserves to be further investigated for use in future breeding programs to improve cow's milk FA-related traits.

Dairy cattle health, wellbeing and productivity are deeply affected by stress. Its influence on metabolism and immune response is well known, but the underlying epigenetic mechanisms require further investigation. In this study, we compared DNA methylation and gene expression signatures between two dairy cattle populations falling in the high- and low-variant tails of the distribution of milk cortisol concentration (MC), a neuroendocrine marker of stress in dairy cows. Reduced Representation Bisulfite Sequencing was used to obtain a methylation map from blood samples of these animals. The high and low groups exhibited similar amounts of methylated CpGs, while we found differences among non-CpG sites. Significant methylation changes were detected in 248 genes. We also identified significant fold differences in the expression of 324 genes. KEGG and Gene Ontology (GO) analysis showed that genes of both groups act together in several pathways, such as nervous system activity, immune regulatory functions and glucocorticoid metabolism. These preliminary results suggest that, in livestock, cortisol secretion could act as a trigger for epigenetic regulation and that peripheral changes in methylation can provide an insight into central nervous system functions.

Hybridisation of wild populations with their domestic counterparts can lead to the loss of wildtype genetic integrity, outbreeding depression, and loss of adaptive features. The Mediterranean island of Sardinia hosts one of the last extant autochthonous European mouflon (Ovis aries musimon) populations. Although conservation policies, including reintroduction plans, have been enforced to preserve Sardinian mouflon, crossbreeding with domestic sheep has been documented. We identified panels of single nucleotide polymorphisms (SNPs) that could act as ancestry informative markers able to assess admixture in feral x domestic sheep hybrids. The medium-density SNP array genotyping data of Sardinian mouflon and domestic sheep (O. aries aries) showing pure ancestry were used as references. We applied a two-step selection algorithm to this data consisting of preselection via Principal Component Analysis followed by a supervised machine learning classification method based on random forest to develop SNP panels of various sizes. We generated ancestry informative marker (AIM) panels and tested their ability to assess admixture in mouflon x domestic sheep hybrids both in simulated and real populations of known ancestry proportions. All the AIM panels recorded high correlations with the ancestry proportion computed using the full medium-density SNP array. The AIM panels proposed here may be used by conservation practitioners as diagnostic tools to exclude hybrids from reintroduction plans and improve conservation strategies for mouflon populations.

In Hong Kong, there is a cattle population of ∼1,200 individuals of uncertain origin and genetic diversity. This population shows heterogeneous morphology, both in body type and pigmentation. Once used as draught animals by the local farmers, they were abandoned around the 1970s due to changes in the economy, and since then have lived as feral populations. To explore the origins of these cattle, we analysed ∼50k genotype data of 21 Hong Kong feral cattle, along with data from 703 individuals of 36 cattle populations of European, African taurine, and Asian origin, the wild x domestic hybrid gayal, plus two wild bovine species, gaur and banteng. To reduce the effect of ascertainment bias ∼4k loci that are polymorphic in the two wild species were selected for further analysis. The stringent SNP selection we applied resulted in increased heterozygosity across all populations studies, compared with the full panel of SNP, thus reducing the impact of ascertainment bias and facilitating the comparison of divergent breeds of cattle. Our results showed that Hong Kong feral cattle have relatively high levels of genetic distinctiveness, possibly due to the low level of artificial selection, and a likely common ancestry with wild species. We found signs of a putative taurine introgression, probably dating to the import of north European breeds during the British colonialism of Hong Kong. We showed that Hong Kong feral cattle, are distinct from Bos taurus and Bos indicus breeds. Our results highlight the distinctiveness of Hong Kong feral cattle and stress the conservation value of this indigenous breed that is likely to harbour adaptive genetic variation, which is a fundamental livestock resource in the face of climate change and diversifying market demands.

Background: In the Neolithic, domestic sheep migrated into Europe and subsequently spread in westerly and northwesterly directions. Reconstruction of these migrations and subsequent genetic events requires a more detailed characterization of the current phylogeographic differentiation. Results: We collected 50 K single nucleotide polymorphism (SNP) profiles of Balkan sheep that are currently found near the major Neolithic point of entry into Europe, and combined these data with published genotypes from southwest-Asian, Mediterranean, central-European and north-European sheep and from Asian and European mouflons. We detected clines, ancestral components and admixture by using variants of common analysis tools: geography-informative supervised principal component analysis (PCA), breed-specific admixture analysis, across-breed $f_{4}$ f 4 profiles and phylogenetic analysis of regional pools of breeds. The regional Balkan sheep populations exhibit considerable genetic overlap, but are clearly distinct from the breeds in surrounding regions. The Asian mouflon did not influence the differentiation of the European domestic sheep and is only distantly related to present-day sheep, including those from Iran where the mouflons were sampled. We demonstrate the occurrence, from southeast to northwest Europe, of a continuously increasing ancestral component of up to 20% contributed by the European mouflon, which is assumed to descend from the original Neolithic domesticates. The overall patterns indicate that the Balkan region and Italy served as post-domestication migration hubs, from which wool sheep reached Spain and north Italy with subsequent migrations northwards. The documented dispersal of Tarentine wool sheep during the Roman period may have been part of this process. Our results also reproduce the documented 18th century admixture of Spanish Merino sheep into several central-European breeds. Conclusions: Our results contribute to a better understanding of the events th

By their paternal transmission, Y-chromosomal haplotypes are sensitive markers of population history and male-mediated introgression. Previous studies identified biallelic single-nucleotide variants in the SRY, ZFY and DDX3Y genes, which in domestic goats identified four major Y-chromosomal haplotypes, Y1A, Y1B, Y2A and Y2B, with a marked geographical partitioning. Here, we extracted goat Y-chromosomal variants from whole-genome sequences of 386 domestic goats (75 breeds) and seven wild goat species, which were generated by the VarGoats goat genome project. Phylogenetic analyses indicated domestic haplogroups corresponding to Y1B, Y2A and Y2B, respectively, whereas Y1A is split into Y1AA and Y1AB. All five haplogroups were detected in 26 ancient DNA samples from southeast Europe or Asia. Haplotypes from present-day bezoars are not shared with domestic goats and are attached to deep nodes of the trees and networks. Haplogroup distributions for 186 domestic breeds indicate ancient paternal population bottlenecks and expansions during migrations into northern Europe, eastern and southern Asia, and Africa south of the Sahara. In addition, sharing of haplogroups indicates male-mediated introgressions, most notably an early gene flow from Asian goats into Madagascar and the crossbreeding that in the 19th century resulted in the popular Boer and Anglo-Nubian breeds. More recent introgressions are those from European goats into the native Korean goat population and from Boer goat into Uganda, Kenya, Tanzania, Malawi and Zimbabwe. This study illustrates the power of the Y-chromosomal variants for reconstructing the history of domestic species with a wide geographical range.

Infectious diseases place an economic burden on aquaculture and a limitation to its growth. An innovative approach to mitigate their impact on production is breeding for disease resistance: selection for domestication, family-based selection, marker-assisted selection, and more recently, genomic selection. Advances in genetics and genomics approaches to the control of infectious diseases are key to increasing aquaculture efficiency, profitability, and sustainability and to reducing its environmental footprint. Interaction and co-evolution between a host and pathogen can, however, turn breeding to boost infectious disease resistance into a potential driver of pathogenic change. Parallel molecular characterization of the pathogen and its virulence and antimicrobial resistance genes is therefore essential to understand pathogen evolution over time in response to host immunity, and to apply appropriate mitigation strategies.

Experimental and epidemiological studies suggest that maternal nutritional status during early pregnancy, including the period around the time of conception, may induce long-lasting epigenetic changes in the offspring. However, this remains largely unexplored in livestock. Therefore, the objective of this study was to evaluate if modification of the maternal diet of sheep (CTR: Control; UND: 50% undernutrition) during the periconceptional period (42 d in total:-14/+28 from mating), would impact CpG methylation in muscle tissue (Longissimus dorsi) of adult offspring (11.5 mo old). Reduced representation bisulfite sequencing identified 262 (Edge-R, FDR < 0.05) and 686 (logistic regression, FDR < 0.001) differentially methylated regions (DMRs) between the UND and CTR groups. Gene ontology analysis identified genes related to development, functions of the muscular system, and steroid hormone receptor activity within the DMRs. The data reported here show that nutritional stress during early pregnancy leads to epigenetic modifications in the muscle of the resulting offspring, with possible implications for cardiac dysfunction, muscle physiology, and meat production. Lay Summary The formation of the epigenetic pattern of an organism is highly sensitive to environmental factors, especially during early mammalian development, when epigenetic reprogramming of the whole genome takes place. In utero adverse conditions experienced during early pregnancy, such as maternal undernutrition, may induce long-lasting epigenetic changes in the resulting offspring. This study investigated the CpG methylation variations in muscle tissue of adult offspring induced by differences in the diet of their mothers during pregnancy. Our data show that undernutrition during pregnancy leads to epigenetic alterations in the muscle of the offspring, with a potential impact on animal health and productivity.