Large-scale analysis of sheep rumen metagenome profiles captured by reduced representation sequencing reveals individual profiles are influenced by the environment and genetics of the host.

BACKGROUND: Producing animal protein while reducing the animal's impact on the environment, e.g., through improved feed efficiency and lowered methane emissions, has gained interest in recent years. Genetic selection is one possible path to reduce the environmental impact of livestock production, but these traits are difficult and expensive to measure on many animals. The rumen microbiome may serve as a proxy for these traits due to its role in feed digestion. Restriction enzyme-reduced representation sequencing (RE-RRS) is a high-throughput and cost-effective approach to rumen metagenome profiling, but the systematic (e.g., sequencing) and biological factors influencing the resulting reference based (RB) and reference free (RF) profiles need to be explored before widespread industry adoption is possible. RESULTS: Metagenome profiles were generated by RE-RRS of 4,479 rumen samples collected from 1,708 sheep, and assigned to eight groups based on diet, age, time off feed, and country (New Zealand or Australia) at the time of sample collection. Systematic effects were found to have minimal influence on metagenome profiles. Diet was a major driver of differences between samples, followed by time off feed, then age of the sheep. The RF approach resulted in more reads being assigned per sample and afforded greater resolution when distinguishing between groups than the RB approach. Normalizing relative abundances within the sampling Cohort abolished structures related to age, diet, and time off feed, allowing a clear signal based on methane emissions to be elucidated. Genus-level abundances of rumen microbes showed low-to-moderate heritability and repeatability and were consistent between diets. CONCLUSIONS: Variation in rumen metagenomic profiles was influenced by diet, age, time off feed and genetics. Not accounting for environmental factors may limit the ability to associate the profile with traits of interest. However, these differences can be accounted for by adjusting for Cohort effects, revealing robust biological signals. The abundances of some genera were consistently heritable and repeatable across different environments, suggesting that metagenomic profiles could be used to predict an individual's future performance, or performance of its offspring, in a range of environments. These results highlight the potential of using rumen metagenomic profiles for selection purposes in a practical, agricultural setting.

Effects of dietary energy density and supplemental rumen undegradable protein on intake, viscera, and carcass composition of lambs recovering from nutritional restriction.

Variation in nutrition is a key determinant of growth, body composition, and the ability of animals to perform to their genetic potential. Depending on the quality of feed available, animals may be able to overcome negative effects of prior nutritional restriction, increasing intake and rates of tissue gain, but full compensation may not occur. A 2 × 3 × 4 factorial serial slaughter study was conducted to examine the effects of prior nutritional restriction, dietary energy density, and supplemental rumen undegradable protein (RUP) on intake, growth, and body composition of lambs. After an initial slaughter (n = 8), 124 4-mo-old Merino cross wethers (28.4 ± 1.8 kg) were assigned to either restricted (LO, 500 g/d) or unrestricted (HI, 1500 g/d) intake of lucerne and oat pellets. After 8 wk, eight lambs/group were slaughtered and tissue weights and chemical composition were measured. Remaining lambs were randomly assigned to a factorial combination of dietary energy density (7.8, 9.2, and 10.7 MJ/kg DM) and supplemental RUP (0, 30, 60, and 90 g/d) and fed ad libitum for a 12- to 13-wk experimental period before slaughter and analysis. By week 3 of the experimental period, lambs fed the same level of energy had similar DMI (g/d) and MEI (MJ/d) (P > 0.05), regardless of prior level of nutrition. Restricted-refed (LO) lambs had higher rates of fat and protein gain than HI lambs (P < 0.05) but had similar visceral masses (P > 0.05). However, LO lambs were lighter and leaner at slaughter, with proportionally larger rumens and livers (P < 0.05). Tissue masses increased with increasing dietary energy density, as did DMI, energy and nitrogen (N) retention (% intake), and rates of protein and fat gain (P < 0.05). The liver increased proportionally with increasing dietary energy density and RUP (P < 0.05), but rumen size decreased relative to the empty body as dietary energy density increased (P < 0.05) and did not respond to RUP (P > 0.05). Fat deposition was greatest in lambs fed 60 g/d supplemental RUP (P < 0.05). However, lambs fed 90 g/d were as lean as lambs that did not receive supplement (P0, P > 0.05), with poorer nitrogen retention and proportionally heavier livers than P0 lambs (P < 0.05). In general, visceral protein was the first tissue to respond to increased intake during refeeding, followed by non-visceral protein and fat, highlighting the influence of differences in tissue response over time on animal performance and body composition.

Animal performance is determined by the combined effects of both prior and current nutrition. The present study used a 2 × 3 × 4 factorial to examine the effects of prior feeding level (HI or LO) on subsequent ad-libitum intake of diets varying in energy density (7.8, 9.2, 10.7 MJ/kg DM) and level of supplemental rumen undegradable protein (RUP; 0, 30, 60, and 90g/d). By week 3 of refeeding, LO and HI lambs had similar feed intake, but LO lambs had proportionally more gut and liver tissue and were lighter and leaner at final slaughter. As dietary energy density increased, the rumen became proportionally smaller while the liver became proportionally larger. Liver size increased with increasing RUP, and lambs fed 30 and 60 g/d were fatter than other lambs. However, lambs fed 90 g/d RUP had less fat than other lambs, as the increased energy requirements of a larger liver and of disposing of excess nitrogen appeared to outweigh any nutritional benefits. Understanding how prior nutrition affects current performance, as well as how tissues vary in their response to the same diet, is key to improving our understanding of animal performance and response to change.

Gene network analysis identifies rumen epithelial cell proliferation, differentiation and metabolic pathways perturbed by diet and correlated with methane production.

Ruminants obtain nutrients from microbial fermentation of plant material, primarily in their rumen, a multilayered forestomach. How the different layers of the rumen wall respond to diet and influence microbial fermentation, and how these process are regulated, is not well understood. Gene expression correlation networks were constructed from full thickness rumen wall transcriptomes of 24 sheep fed two different amounts and qualities of a forage and measured for methane production. The network contained two major negatively correlated gene sub-networks predominantly representing the epithelial and muscle layers of the rumen wall. Within the epithelium sub-network gene clusters representing lipid/oxo-acid metabolism, general metabolism and proliferating and differentiating cells were identified. The expression of cell cycle and metabolic genes was positively correlated with dry matter intake, ruminal short chain fatty acid concentrations and methane production. A weak correlation between lipid/oxo-acid metabolism genes and methane yield was observed. Feed consumption level explained the majority of gene expression variation, particularly for the cell cycle genes. Many known stratified epithelium transcription factors had significantly enriched targets in the epithelial gene clusters. The expression patterns of the transcription factors and their targets in proliferating and differentiating skin is mirrored in the rumen, suggesting conservation of regulatory systems.

Explaining the variation in lamb longissimus shear force across and within ageing periods using protein degradation, sarcomere length and collagen characteristics.

Meat tenderness is known to be affected by sarcomere length (SL), proteolysis and collagen content (CC). Sixty lambs were slaughtered and the Longissimus muscle was sampled. Samples for shear force (SF), SL, proteolysis indicators (desmin degradation, particle size: PS) and CC were taken after the allotted ageing periods (1, 7, and 14 days). PS explained a large part of the variation in shear force (approximately 34%) when modelled across ageing periods. Other factors (CC, SL) combined with proteolysis indicators (PS, desmin degradation) explained just under 40% of the variation in shear force. Within ageing periods SL explained a small, but significant, part of the variation in shear force after 14 days of ageing (8%) and at day 1 of ageing desmin degradation explained 17% of the variation in shear force. Methods to improve the tenderness of lamb longissimus muscle should focus on increasing the extent of post-mortem proteolysis, when processing conditions are sufficient to prevent muscle fibre shortening.

Low-methane yield sheep have smaller rumens and shorter rumen retention time.

In the present study, following the measurement of methane emissions from 160 mature ewes three times, a subset of twenty ewes was selected for further emission and physiological studies. Ewes were selected on the basis of methane yield (MY; g CH4/kg DM intake) being low (Low MY: >1 sd below the mean; n 10) or high (High MY: >1 sd above the mean; n 10) when fed a blended chaff ration at a fixed feeding level (1·2-fold maintenance energy requirements). The difference between the Low- and High-MY groups observed at the time of selection was maintained (P= 0·001) when remeasured 1-7 months later during digesta kinetics studies. Low MY was associated with a shorter mean retention time of particulate (P< 0·01) and liquid (P< 0·001) digesta, less amounts of rumen particulate contents (P< 0·01) and a smaller rumen volume (P< 0·05), but not apparent DM digestibility (P= 0·27) or urinary allantoin excretion (P= 0·89). Computer tomography scanning of the sheep's rumens after an overnight fast revealed a trend towards the Low-MY sheep having more clearly demarcated rumen gas and liquid phases (P= 0·10). These findings indicate that the selection of ruminants for low MY may have important consequences for an animal's nutritional physiology.

A genome wide survey of SNP variation reveals the genetic structure of sheep breeds.

The genetic structure of sheep reflects their domestication and subsequent formation into discrete breeds. Understanding genetic structure is essential for achieving genetic improvement through genome-wide association studies, genomic selection and the dissection of quantitative traits. After identifying the first genome-wide set of SNP for sheep, we report on levels of genetic variability both within and between a diverse sample of ovine populations. Then, using cluster analysis and the partitioning of genetic variation, we demonstrate sheep are characterised by weak phylogeographic structure, overlapping genetic similarity and generally low differentiation which is consistent with their short evolutionary history. The degree of population substructure was, however, sufficient to cluster individuals based on geographic origin and known breed history. Specifically, African and Asian populations clustered separately from breeds of European origin sampled from Australia, New Zealand, Europe and North America. Furthermore, we demonstrate the presence of stratification within some, but not all, ovine breeds. The results emphasize that careful documentation of genetic structure will be an essential prerequisite when mapping the genetic basis of complex traits. Furthermore, the identification of a subset of SNP able to assign individuals into broad groupings demonstrates even a small panel of markers may be suitable for applications such as traceability.

Evidence for multiple alleles effecting muscling and fatness at the ovine GDF8 locus.

BACKGROUND: The current investigation surveyed genetic polymorphism at the ovine GDF8 locus and determined its contribution to variation in muscling and fatness in sheep. RESULTS: Re-sequencing 2988 bp from a panel of 15 sires revealed a total of six SNP, none of which were located within exons of the gene. One of the identified SNP, g+6723G>A, is known to increase muscularity within the Belgian Texel. A genetic survey of 326 animals revealed that the mutation is near fixation within Australian Texels and present in additional breeds including White Suffolk, Poll Dorset and Lincoln. Using a resource population comprising 15 sires and 1191 half-sib progeny with genotypic data, the effect of this and other SNP was tested against a set of 50 traits describing growth, muscling, fatness, yield, meat and eating quality. The loss of function allele (g+6723A) showed significant effects on slaughter measurements of muscling and fatness. No effect was detected on objectively assessed meat quality however evidence was found for an association between g+6723G>A, decreased intramuscular fat and reduced eating quality. Haplotype analysis using flanking microsatellites was performed to search for evidence of currently unidentified mutations which might affect production traits. Four haplotypes were identified that do not carry g+6723A but which showed significant associations with muscling and fatness. CONCLUSION: The finding that g+6723G>A is present within Australian sheep facilitated an independent evaluation into its phenotypic consequence. Testing was conducted using a separate genetic background and animals raised in different environments to the Belgian Texel in which it was first identified. The observation that the direction and size of effects for g+6723A is approximately consistent represented a robust validation of the effects of the mutation. Based on observed allele frequencies within breeds, selection for g+6723A will have the largest impact within the White Suffolk. GDF8 may harbour additional mutations which serve to influence economically important traits in sheep.

Using comparative genomics to reorder the human genome sequence into a virtual sheep genome.

BACKGROUND: Is it possible to construct an accurate and detailed subgene-level map of a genome using bacterial artificial chromosome (BAC) end sequences, a sparse marker map, and the sequences of other genomes? RESULTS: A sheep BAC library, CHORI-243, was constructed and the BAC end sequences were determined and mapped with high sensitivity and low specificity onto the frameworks of the human, dog, and cow genomes. To maximize genome coverage, the coordinates of all BAC end sequence hits to the cow and dog genomes were also converted to the equivalent human genome coordinates. The 84,624 sheep BACs (about 5.4-fold genome coverage) with paired ends in the correct orientation (tail-to-tail) and spacing, combined with information from sheep BAC comparative genome contigs (CGCs) built separately on the dog and cow genomes, were used to construct 1,172 sheep BAC-CGCs, covering 91.2% of the human genome. Clustered non-tail-to-tail and outsize BACs located close to the ends of many BAC-CGCs linked BAC-CGCs covering about 70% of the genome to at least one other BAC-CGC on the same chromosome. Using the BAC-CGCs, the intrachromosomal and interchromosomal BAC-CGC linkage information, human/cow and vertebrate synteny, and the sheep marker map, a virtual sheep genome was constructed. To identify BACs potentially located in gaps between BAC-CGCs, an additional set of 55,668 sheep BACs were positioned on the sheep genome with lower confidence. A coordinate conversion process allowed us to transfer human genes and other genome features to the virtual sheep genome to display on a sheep genome browser. CONCLUSION: We demonstrate that limited sequencing of BACs combined with positioning on a well assembled genome and integrating locations from other less well assembled genomes can yield extensive, detailed subgene-level maps of mammalian genomes, for which genomic resources are currently limited.