De Novo Long-Read Whole-Genome Assemblies and the Comparative Pan-Genome Analysis of Ascochyta Blight Pathogens Affecting Field Pea.

Ascochyta Blight (AB) is a major disease of many cool-season legumes globally. In field pea, three fungal pathogens have been identified to be responsible for this disease in Australia, namely Peyronellaea pinodes, Peyronellaea pinodella and Phoma koolunga. Limited genomic resources for these pathogens have been generated, which has hampered the implementation of effective management strategies and breeding for resistant cultivars. Using Oxford Nanopore long-read sequencing, we report the first high-quality, fully annotated, near-chromosome-level nuclear and mitochondrial genome assemblies for 18 isolates from the Australian AB complex. Comparative genome analysis was performed to elucidate the differences and similarities between species and isolates using phylogenetic relationships and functional diversity. Our data indicated that P. pinodella and P. koolunga are heterothallic, while P. pinodes is homothallic. More homology and orthologous gene clusters are shared between P. pinodes and P. pinodella compared to P. koolunga. The analysis of the repetitive DNA content showed differences in the transposable repeat composition in the genomes and their expression in the transcriptomes. Significant repeat expansion in P. koolunga's genome was seen, with strong repeat-induced point mutation (RIP) activity being evident. Phylogenetic analysis revealed that genetic diversity can be exploited for species marker development. This study provided the much-needed genetic resources and characterization of the AB species to further drive research in key areas such as disease epidemiology and host-pathogen interactions.

Improving Genomic Selection for Heat Tolerance in Dairy Cattle: Current Opportunities and Future Directions.

Heat tolerance is the ability of an animal to maintain production and reproduction levels under hot and humid conditions and is now a trait of economic relevance in dairy systems worldwide because of an escalating warming climate. The Australian dairy population is one of the excellent study models for enhancing our understanding of the biology of heat tolerance because they are predominantly kept outdoors on pastures where they experience direct effects of weather elements (e.g., solar radiation). In this article, we focus on evidence from recent studies in Australia that leveraged large a dataset [∼40,000 animals with phenotypes and 15 million whole-genome sequence variants] to elucidate the genetic basis of thermal stress as a critical part of the strategy to breed cattle adapted to warmer environments. Genotype-by-environment interaction (i.e., G × E) due to temperature and humidity variation is increasing, meaning animals are becoming less adapted (i.e., more sensitive) to changing environments. There are opportunities to reverse this trend and accelerate adaptation to warming climate by 1) selecting robust or heat-resilient animals and 2) including resilience indicators in breeding goals. Candidate causal variants related to the nervous system and metabolic functions are relevant for heat tolerance and, therefore, key for improving this trait. This could include adding these variants in the custom SNP panels used for routine genomic evaluations or as the basis to design specific agonist or antagonist compounds for lowering core body temperature under heat stress conditions. Indeed, it was encouraging to see that adding prioritized functionally relevant variants into the 50k SNP panel (i.e., the industry panel used for genomic evaluation in Australia) increased the prediction accuracy of heat tolerance by up to 10% units. This gain in accuracy is critical because genetic improvement has a linear relationship with prediction accuracy. Overall, while this article used data mainly from Australia, this could benefit other countries that aim to develop breeding values for heat tolerance, considering that the warming climate is becoming a topical issue worldwide.

Dietary wheat and reduced methane yield are linked to rumen microbiome changes in dairy cows.

Fermentation of pasture grasses and grains in the rumen of dairy cows and other ruminants produces methane as a by-product, wasting energy and contributing to the atmospheric load of greenhouse gasses. Many feeding trials in farmed ruminants have tested the impact of dietary components on feed efficiency, productivity and methane yield (MeY). Such diets remodel the rumen microbiome, altering bacterial, archaeal, fungal and protozoan populations, with an altered fermentation outcome. In dairy cows, some dietary grains can reduce enteric methane production. This is especially true of wheat, in comparison to corn or barley. Using a feeding trial of cows fed rolled wheat, corn or barley grain, in combination with hay and canola, we identified wheat-associated changes in the ruminal microbiome. Ruminal methane production, pH and VFA concentration data together with 16S rRNA gene amplicon sequences were used to compare ruminal bacterial and archaeal populations across diets. Differential abundance analysis of clustered sequences (OTU) identified members of the bacterial families Lachnospiraceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Selenomonadaceae, Anaerovoracaceae and Fibrobacteraceae having a strong preference for growth in wheat-fed cows. Within the methanogenic archaea, (at >99% 16S rRNA sequence identity) the growth of Methanobrevibacter millerae was favoured by the non-wheat diets, while Methanobrevibacter olleyae was unaffected. From the wheat-preferring bacteria, correlation analysis found OTU strongly linked to reduced MeY, reduced pH and raised propionic acid levels. OTU from the genera Shuttleworthia and Prevotella_7 and especially Selenomonadaceae had high anti-methane correlations. An OTU likely representing (100% sequence identity) the fumarate-reducing, hydrogen-utilising, rumen bacterium Mitsuokella jalaludinii, had an especially high negative correlation coefficient (-0.83) versus MeY and moderate correlation (-0.6) with rumen pH, strongly suggesting much of the MeY suppression is due to reduced hydrogen availablity. Other OTU, representing as yet unknown species from the Selenomonadaceae family and the genera Prevotella_7, Fibrobacter and Syntrophococcus also had high to moderate negative MeY correlations, but low correlation with pH. These latter likely represent bacterial species able to reduce MeY without causing greater ruminal acidity, making them excellent candidates, provided they can be isolated, for development as anti-methane probiotics.

Recovery of mitogenomes from whole genome sequences to infer maternal diversity in 1883 modern taurine and indicine cattle.

Maternal diversity based on a sub-region of mitochondrial genome or variants were commonly used to understand past demographic events in livestock. Additionally, there is growing evidence of direct association of mitochondrial genetic variants with a range of phenotypes. Therefore, this study used complete bovine mitogenomes from a large sequence database to explore the full spectrum of maternal diversity. Mitogenome diversity was evaluated among 1883 animals representing 156 globally important cattle breeds. Overall, the mitogenomes were diverse: presenting 11 major haplogroups, expanding to 1309 unique haplotypes, with nucleotide diversity 0.011 and haplotype diversity 0.999. A small proportion of African taurine (3.5%) and indicine (1.3%) haplogroups were found among the European taurine breeds and composites. The haplogrouping was largely consistent with the population structure derived from alternate clustering methods (e.g. PCA and hierarchical clustering). Further, we present evidence confirming a new indicine subgroup (I1a, 64 animals) mainly consisting of breeds originating from China and characterised by two private mutations within the I1 haplogroup. The total genetic variation was attributed mainly to within-breed variance (96.9%). The accuracy of the imputation of missing genotypes was high (99.8%) except for the relatively rare heteroplasmic genotypes, suggesting the potential for trait association studies within a breed.

Functionally prioritised whole-genome sequence variants improve the accuracy of genomic prediction for heat tolerance.

BACKGROUND: Heat tolerance is a trait of economic importance in the context of warm climates and the effects of global warming on livestock production, reproduction, health, and well-being. This study investigated the improvement in prediction accuracy for heat tolerance when selected sets of sequence variants from a large genome-wide association study (GWAS) were combined with a standard 50k single nucleotide polymorphism (SNP) panel used by the dairy industry. METHODS: Over 40,000 dairy cattle with genotype and phenotype data were analysed. The phenotypes used to measure an individual's heat tolerance were defined as the rate of decline in milk production traits with rising temperature and humidity. We used Holstein and Jersey cows to select sequence variants linked to heat tolerance. The prioritised sequence variants were the most significant SNPs passing a GWAS p-value threshold selected based on sliding 100-kb windows along each chromosome. We used a bull reference set to develop the genomic prediction equations, which were then validated in an independent set of Holstein, Jersey, and crossbred cows. Prediction analyses were performed using the BayesR, BayesRC, and GBLUP methods. RESULTS: The accuracy of genomic prediction for heat tolerance improved by up to 0.07, 0.05, and 0.10 units in Holstein, Jersey, and crossbred cows, respectively, when sets of selected sequence markers from Holstein cows were added to the 50k SNP panel. However, in some scenarios, the prediction accuracy decreased unexpectedly with the largest drop of - 0.10 units for the heat tolerance fat yield trait observed in Jersey cows when 50k plus pre-selected SNPs from Holstein cows were used. Using pre-selected SNPs discovered on a combined set of Holstein and Jersey cows generally improved the accuracy, especially in the Jersey validation. In addition, combining Holstein and Jersey bulls in the reference set generally improved prediction accuracy in most scenarios compared to using only Holstein bulls as the reference set. CONCLUSIONS: Informative sequence markers can be prioritised to improve the genomic prediction of heat tolerance in different breeds. In addition to providing biological insight, these variants could also have a direct application for developing customized SNP arrays or can be used via imputation in current industry SNP panels.

New loci and neuronal pathways for resilience to heat stress in cattle.

While understanding the genetic basis of heat tolerance is crucial in the context of global warming's effect on humans, livestock, and wildlife, the specific genetic variants and biological features that confer thermotolerance in animals are still not well characterized. We used dairy cows as a model to study heat tolerance because they are lactating, and therefore often prone to thermal stress. The data comprised almost 0.5 million milk records (milk, fat, and proteins) of 29,107 Australian Holsteins, each having around 15 million imputed sequence variants. Dairy animals often reduce their milk production when temperature and humidity rise; thus, the phenotypes used to measure an individual's heat tolerance were defined as the rate of milk production decline (slope traits) with a rising temperature-humidity index. With these slope traits, we performed a genome-wide association study (GWAS) using different approaches, including conditional analyses, to correct for the relationship between heat tolerance and level of milk production. The results revealed multiple novel loci for heat tolerance, including 61 potential functional variants at sites highly conserved across 100 vertebrate species. Moreover, it was interesting that specific candidate variants and genes are related to the neuronal system (ITPR1, ITPR2, and GRIA4) and neuroactive ligand-receptor interaction functions for heat tolerance (NPFFR2, CALCR, and GHR), providing a novel insight that can help to develop genetic and management approaches to combat heat stress.

Mitochondrial protein gene expression and the oxidative phosphorylation pathway associated with feed efficiency and energy balance in dairy cattle.

Feed efficiency and energy balance are important traits underpinning profitability and environmental sustainability in animal production. They are complex traits, and our understanding of their underlying biology is currently limited. One measure of feed efficiency is residual feed intake (RFI), which is the difference between actual and predicted intake. Variation in RFI among individuals is attributable to the metabolic efficiency of energy utilization. High RFI (H_RFI) animals require more energy per unit of weight gain or milk produced compared with low RFI (L_RFI) animals. Energy balance (EB) is a closely related trait calculated very similarly to RFI. Cellular energy metabolism in mitochondria involves mitochondrial protein (MiP) encoded by both nuclear (NuMiP) and mitochondrial (MtMiP) genomes. We hypothesized that MiP genes are differentially expressed (DE) between H_RFI and L_RFI animal groups and similarly between negative and positive EB groups. Our study aimed to characterize MiP gene expression in white blood cells of H_RFI and L_RFI cows using RNA sequencing to identify genes and biological pathways associated with feed efficiency in dairy cattle. We used the top and bottom 14 cows ranked for RFI and EB out of 109 animals as H_RFI and L_RFI, and positive and negative EB groups, respectively. The gene expression counts across all nuclear and mitochondrial genes for animals in each group were used for differential gene expression analyses, weighted gene correlation network analysis, functional enrichment, and identification of hub genes. Out of 244 DE genes between RFI groups, 38 were MiP genes. The DE genes were enriched for the oxidative phosphorylation (OXPHOS) and ribosome pathways. The DE MiP genes were underexpressed in L_RFI (and negative EB) compared with the H_RFI (and positive EB) groups, suggestive of reduced mitochondrial activity in the L_RFI group. None of the MtMiP genes were among the DE MiP genes between the groups, which suggests a non-rate limiting role of MtMiP genes in feed efficiency and warrants further investigation. The role of MiP, particularly the NuMiP and OXPHOS pathways in RFI, was also supported by our gene correlation network analysis and the hub gene identification. We validated the findings in an independent data set. Overall, our study suggested that differences in feed efficiency in dairy cows may be linked to differences in cellular energy demand. This study broadens our knowledge of the biology of feed efficiency in dairy cattle.

Fungus-originated genes in the genomes of cereal and pasture grasses acquired through ancient lateral transfer.

Evidence for ancestral gene transfer between Epichloë fungal endophyte ancestors and their host grass species is described. From genomes of cool-season grasses (the Poeae tribe), two Epichloë-originated genes were identified through DNA sequence similarity analysis. The two genes showed 96% and 85% DNA sequence identities between the corresponding Epichloë genes. One of the genes was specific to the Loliinae sub-tribe. The other gene was more widely conserved in the Poeae and Triticeae tribes, including wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). The genes were independently transferred during the last 39 million years. The transferred genes were expressed in plant tissues, presumably retaining molecular functions. Multiple gene transfer events between the specific plant and fungal lineages are unique. A range of cereal crops is included in the Poeae and Triticeae tribes, and the Loliinae sub-tribe is consisted of economically important pasture and forage crops. Identification and characterisation of the 'natural' adaptation transgenes in the genomes of cereals, and pasture and forage grasses, that worldwide underpin the production of major foods, such as bread, meat, and milk, may change the 'unnatural' perception status of transgenic and gene-edited plants.

Expression of mitochondrial protein genes encoded by nuclear and mitochondrial genomes correlate with energy metabolism in dairy cattle.

BACKGROUND: Mutations in the mitochondrial genome have been implicated in mitochondrial disease, often characterized by impaired cellular energy metabolism. Cellular energy metabolism in mitochondria involves mitochondrial proteins (MP) from both the nuclear (NuMP) and mitochondrial (MtMP) genomes. The expression of MP genes in tissues may be tissue specific to meet varying specific energy demands across the tissues. Currently, the characteristics of MP gene expression in tissues of dairy cattle are not well understood. In this study, we profile the expression of MP genes in 29 adult and six foetal tissues in dairy cattle using RNA sequencing and gene expression analyses: particularly differential gene expression and co-expression network analyses. RESULTS: MP genes were differentially expressed (DE; over-expressed or under-expressed) across tissues in cattle. All 29 tissues showed DE NuMP genes in varying proportions of over-expression and under-expression. On the other hand, DE of MtMP genes was observed in < 50% of tissues and notably MtMP genes within a tissue was either all over-expressed or all under-expressed. A high proportion of NuMP (up to 60%) and MtMP (up to 100%) genes were over-expressed in tissues with expected high metabolic demand; heart, skeletal muscles and tongue, and under-expressed (up to 45% of NuMP, 77% of MtMP genes) in tissues with expected low metabolic rates; leukocytes, thymus, and lymph nodes. These tissues also invariably had the expression of all MtMP genes in the direction of dominant NuMP genes expression. The NuMP and MtMP genes were highly co-expressed across tissues and co-expression of genes in a cluster were non-random and functionally enriched for energy generation pathway. The differential gene expression and co-expression patterns were validated in independent cow and sheep datasets. CONCLUSIONS: The results of this study support the concept that there are biological interaction of MP genes from the mitochondrial and nuclear genomes given their over-expression in tissues with high energy demand and co-expression in tissues. This highlights the importance of considering MP genes from both genomes in future studies related to mitochondrial functions and traits related to energy metabolism.

Faecalibacterium diversity in dairy cow milk.

The bacterial species, Faecalibacterium prausnitzii, beneficial to humans and animals and found in mammalian and avian gut, is also occasionally found in dairy cow milk. It is one of the butyrate-producing bacteria of the colon, has anti-inflammatory properties and its abundance in the gut is negatively correlated with obesity in humans. Several strains differing in their functional capability, have been identified. It is important therefore, milk being a potential source of F. prausnitzii as a novel probiotic, to investigate the diversity of this species in bovine milk. Using 16s rRNA gene amplicons we find 292 different dereplicated Faecalibacterium-related amplicons in a herd of 21 dairy cows. The distribution of the 20 most abundant amplicons with >97% identity to a Greengenes OTU varies from cow to cow. Clustering of the 292 pooled sequences from all cows at 99.6% identity finds 4 likely Faecalibacterium phylotypes with >98.5% identity to an F. prausnitzii reference sequence. Sequence alignment and phylogenetic analysis shows these phylotypes are distinct from 34 other species from the Ruminococcaceae family and displaying the sequence clusters as a network illustrates how each cluster is composed of sequences from multiple cows. We conclude there are several phylotypes of Faecalibacterium prausnitzii (the only species so far defined for the genus) in this dairy herd with cows being inoculated with a mixture of several strains from a common source. We conclude that not only can Faecalibacterium be detected in dairy cow milk (as noted by others) but that there exist multiple different strains in the milk of a dairy herd. Therefore milk, as an alternative to faeces, offers the opportunity of discovering new strains with potential probiotic application.

Fine-mapping sequence mutations with a major effect on oligosaccharide content in bovine milk.

Human milk contains abundant oligosaccharides (OS) which are believed to have strong health benefits for neonates. OS are a minor component of bovine milk and little is known about how the production of OS is regulated in the bovine mammary gland. We have measured the abundance of 12 major OS in milk of 360 cows, which had high density SNP marker genotypes. Most of the OS were found to be highly heritable (h2 between 50 and 84%). A genome-wide association study allowed us to fine-map several QTL and identify candidate genes with major effects on five OS. Among them, a putative causal mutation close to the ABO gene on Chromosome 11 accounted for approximately 80% of genetic variance for two OS, N-acetylgalactosaminyllactose and lacto-N-neotetraose. This mutation lies very close to a variant associated with the expression levels of ABO. A third QTL mapped close to ST3GAL6 on Chromosome 1 explaining 33% of genetic variation of an abundant OS, 3'-sialyllactose. The presence of major gene effects suggests that targeted marker-assisted selection would lead to a significant increase in the level of these OS in milk. This is the first attempt to map candidate genes and causal mutations for bovine milk OS.

Putative bovine topological association domains and CTCF binding motifs can reduce the search space for causative regulatory variants of complex traits.

BACKGROUND: Topological association domains (TADs) are chromosomal domains characterised by frequent internal DNA-DNA interactions. The transcription factor CTCF binds to conserved DNA sequence patterns called CTCF binding motifs to either prohibit or facilitate chromosomal interactions. TADs and CTCF binding motifs control gene expression, but they are not yet well defined in the bovine genome. In this paper, we sought to improve the annotation of bovine TADs and CTCF binding motifs, and assess whether the new annotation can reduce the search space for cis-regulatory variants. RESULTS: We used genomic synteny to map TADs and CTCF binding motifs from humans, mice, dogs and macaques to the bovine genome. We found that our mapped TADs exhibited the same hallmark properties of those sourced from experimental data, such as housekeeping genes, transfer RNA genes, CTCF binding motifs, short interspersed elements, H3K4me3 and H3K27ac. We showed that runs of genes with the same pattern of allele-specific expression (ASE) (either favouring paternal or maternal allele) were often located in the same TAD or between the same conserved CTCF binding motifs. Analyses of variance showed that when averaged across all bovine tissues tested, TADs explained 14% of ASE variation (standard deviation, SD: 0.056), while CTCF explained 27% (SD: 0.078). Furthermore, we showed that the quantitative trait loci (QTLs) associated with gene expression variation (eQTLs) or ASE variation (aseQTLs), which were identified from mRNA transcripts from 141 lactating cows' white blood and milk cells, were highly enriched at putative bovine CTCF binding motifs. The linearly-furthermost, and most-significant aseQTL and eQTL for each genic target were located within the same TAD as the gene more often than expected (Chi-Squared test P-value < 0.001). CONCLUSIONS: Our results suggest that genomic synteny can be used to functionally annotate conserved transcriptional components, and provides a tool to reduce the search space for causative regulatory variants in the bovine genome.

Seasonal variation of polar lipid content in bovine milk.

Seasonal change of milk composition could offer opportunities for dairy manufacturers. A systematic survey on seasonal variation of six classes of polar lipids was conducted with 19 Holstein cows over the entire milking season using liquid chromatography-mass spectrometry technique. This study revealed that most polar lipid classes were positively correlated with the total fat content, but negatively correlated with fat globule size. All polar lipid classes displayed a large cow-to-cow variation as well as seasonal variation. All of the six classes showed a gradual increase over the milking season with the highest concentration observed in May (autumn). However, the proportion of different polar lipid classes remained constant during the entire milking season. This finding suggests that the production of polar lipids is highly regulated in the mammary gland. The implication of such a seasonal variation of polar lipids in the nutritional and technological aspects of milk is discussed.

Horizontal transfer of a ß-1,6-glucanase gene from an ancestral species of fungal endophyte to a cool-season grass host.

Molecular characterisation has convincingly demonstrated some types of horizontal gene transfer in eukaryotes, but nuclear gene transfer between distantly related eukaryotic groups appears to have been rare. For angiosperms (flowering plants), nuclear gene transfer events identified to date have been confined to genes originating from prokaryotes or other plant species. In this report, evidence for ancient horizontal transfer of a fungal nuclear gene, encoding a ß-1,6-glucanase enzyme for fungal cell wall degradation, into an angiosperm lineage is presented for the first time. The gene was identified from de novo sequencing and assembly of the genome and transcriptome of perennial ryegrass, a cool-season grass species. Molecular analysis confirmed the presence of the complete gene in the genome of perennial ryegrass. No corresponding sequence was found in other plant species, apart from members of the Poeae sub-tribes Loliinae and Dactylidinae. Evidence suggests that a common ancestor of the two sub-tribes acquired the gene from a species ancestral to contemporary grass-associated fungal endophytes around 9-13 million years ago. This first report of horizontal transfer of a nuclear gene from a taxonomically distant eukaryote to modern flowering plants provides evidence for a novel adaptation mechanism in angiosperms.

Putative enhancer sites in the bovine genome are enriched with variants affecting complex traits.

BACKGROUND: Enhancers are non-coding DNA sequences, which when they are bound by specific proteins increase the level of gene transcription. Enhancers activate unique gene expression patterns within cells of different types or under different conditions. Enhancers are key contributors to gene regulation, and causative variants that affect quantitative traits in humans and mice have been located in enhancer regions. However, in the bovine genome, enhancers as well as other regulatory elements are not yet well defined. In this paper, we sought to improve the annotation of bovine enhancer regions by using publicly available mammalian enhancer information. To test if the identified putative bovine enhancer regions are enriched with functional variants that affect milk production traits, we performed genome-wide association studies using imputed whole-genome sequence data followed by meta-analysis and enrichment analysis. RESULTS: We produced a library of candidate bovine enhancer regions by using publicly available bovine ChIP-Seq enhancer data in combination with enhancer data that were identified based on sequence homology with human and mouse enhancer databases. We found that imputed whole-genome sequence variants associated with milk production traits in 16,581 dairy cattle were enriched with enhancer regions that were marked by bovine-liver H3K4me3 and H3K27ac histone modifications from both permutation tests and gene set enrichment analysis. Enhancer regions that were identified based on sequence homology with human and mouse enhancer regions were not as strongly enriched with trait-associated sequence variants as the bovine ChIP-Seq candidate enhancer regions. The bovine ChIP-Seq enriched enhancer regions were located near genes and quantitative trait loci that are associated with pregnancy, growth, disease resistance, meat quality and quantity, and milk quality and quantity traits in dairy and beef cattle. CONCLUSIONS: Our results suggest that sequence variants within enhancer regions that are located in bovine non-coding genomic regions contribute to the variation in complex traits. The level of enrichment was higher in bovine-specific enhancer regions that were identified by detecting histone modifications H3K4me3 and H3K27ac in bovine liver tissues than in enhancer regions identified by sequence homology with human and mouse data. These results highlight the need to use bovine-specific experimental data for the identification of enhancer regions.

Seasonal Variation of Triacylglycerol Profile of Bovine Milk.

Milk contains 3-6% of fat, of which the dominant component is triacylglycerol (TAG). Over 100 TAG groups can be readily detected in any non-enriched milk sample by LC-MS; most TAG groups contain several isomers (TAG molecules with different fatty acid composition), which cannot be fully resolved chromatographically by any single stationary phase. TAG profile of mature milk from 19 cows was surveyed in this study for eight consecutive months using RP-LC-Orbitrap MS. It was found that TAG profile of milk was not constant throughout the milking season and the seasonal pattern varied with TAG groups. The overall unsaturation level of TAG was stable from October 2013 to January 2014, decreased in February/March 2014 and then increased from April and peaked in May 2014. In addition to the seasonal fluctuation in TAG profile, the proportion of different isomeric species within a TAG group also changed substantially across seasons. However, the proportion of different positional isomers within a given TAG group does not seem to vary during the milking season. To our knowledge, this is the first report on the seasonal change of milk lipid at the TAG group and isomer level.

Optimization of a single phase method for lipid extraction from milk.

For LC-MS-based lipidomic analysis of milk, total lipid extraction from raw milk is generally conducted with Folch or Bligh and Dyer methods; both methods are based on two-phase partition of lipids, and thus time-consuming. In this work, three solvent systems for one-phase extraction of milk lipids were compared with the standard Folch method. Two of the solvent systems (butanol/methanol, 3:1 and 1:1) previously tested for lipid extraction from plasma were found to provide adequate extraction for polar lipids, but incomplete extraction for triglycerides, especially highly lipophilic species. By contrast, our newly designed solvent mix composed of butanol, methanol and chloroform (at a 3:5:4 ratio) provided similar extraction efficiency for triglycerides and higher yield for some of the phospholipids, as compared to the Folch method. This new one-phase extraction method is very simple yet comprehensive and thus suitable for high throughput lipid analysis of milk samples.

Comparison of Molecular Species Distribution of DHA-Containing Triacylglycerols in Milk and Different Infant Formulas by Liquid Chromatography-Mass Spectrometry.

Long-chain polyunsaturated fatty acids (LC-PUFA) are an important nutritional lipid and have potential in being able to promote human health. Docosahexaenoic acid (DHA, C22:6ω3) is often added in infant formulas to meet the nutritional requirement of formula-fed infants. A comprehensive survey on DHA-containing triacylglycerol (DHA-TAG) molecular species has been conducted for seven infant formulas (IFs) sourced from Australia, Europe, and the USA as well as bovine milk and human milk. Using LC-triple quadrupole MS and LC-LTQ-orbitrap MS we were able to identify and quantify 56 DHA-TAG species in these samples; the fatty acid structure of these species was assigned using their MS(2) spectra. The species composition of DHA-TAG was found to be different between bovine milk, human milk, and IFs and also between different brands of IFs. Bovine milk and human milk contain DHA-TAG of smaller molecular size (728-952 Da), whereas five out of the seven IF samples contain species of broader mass range (from 728 to 1035 Da). Our study indicates that two types of DHA were used in the seven IF products surveyed and that there is very large difference in molecular species distribution in different IF products that may influence the fine nutritional profile and biological functions of IF products.

Accounting for dominance to improve genomic evaluations of dairy cows for fertility and milk production traits.

BACKGROUND: Dominance effects may contribute to genetic variation of complex traits in dairy cattle, especially for traits closely related to fitness such as fertility. However, traditional genetic evaluations generally ignore dominance effects and consider additive genetic effects only. Availability of dense single nucleotide polymorphisms (SNPs) panels provides the opportunity to investigate the role of dominance in quantitative variation of complex traits at both the SNP and animal levels. Including dominance effects in the genomic evaluation of animals could also help to increase the accuracy of prediction of future phenotypes. In this study, we estimated additive and dominance variance components for fertility and milk production traits of genotyped Holstein and Jersey cows in Australia. The predictive abilities of a model that accounts for additive effects only (additive), and a model that accounts for both additive and dominance effects (additive + dominance) were compared in a fivefold cross-validation. RESULTS: Estimates of the proportion of dominance variation relative to phenotypic variation that is captured by SNPs, for production traits, were up to 3.8 and 7.1 % in Holstein and Jersey cows, respectively, whereas, for fertility, they were equal to 1.2 % in Holstein and very close to zero in Jersey cows. We found that including dominance in the model was not consistently advantageous. Based on maximum likelihood ratio tests, the additive + dominance model fitted the data better than the additive model, for milk, fat and protein yields in both breeds. However, regarding the prediction of phenotypes assessed with fivefold cross-validation, including dominance effects in the model improved accuracy only for fat yield in Holstein cows. Regression coefficients of phenotypes on genetic values and mean squared errors of predictions showed that the predictive ability of the additive + dominance model was superior to that of the additive model for some of the traits. CONCLUSIONS: In both breeds, dominance effects were significant (P < 0.01) for all milk production traits but not for fertility. Accuracy of prediction of phenotypes was slightly increased by including dominance effects in the genomic evaluation model. Thus, it can help to better identify highly performing individuals and be useful for culling decisions.