Dietary Fermentation Product of Aspergillus Oryzae Prevents Increases in Gastrointestinal Permeability ('Leaky Gut') in Horses Undergoing Combined Transport and Exercise.

Equine leaky gut syndrome is characterized by gastrointestinal hyperpermeability and may be associated with adverse health effects in horses. The purpose was to evaluate the effects of a prebiotic Aspergillus oryzae product (SUPP) on stress-induced gastrointestinal hyperpermeability. Eight horses received a diet containing SUPP (0.02 g/kg BW) or an unsupplemented diet (CO) (n = 4 per group) for 28 days. On Days 0 and 28, horses were intubated with an indigestible marker of gastrointestinal permeability (iohexol). Half the horses from each feeding group underwent 60 min of transport by trailer immediately followed by a moderate-intensity exercise bout of 30 min (EX), and the remaining horses stayed in stalls as controls (SED). Blood was sampled before iohexol, immediately after trailering, and at 0, 1, 2, 4, and 8 h post-exercise. At the end of the feeding period, horses were washed out for 28 days before being assigned to the opposite feeding group, and the study was replicated. Blood was analyzed for iohexol (HPLC), lipopolysaccharide (ELISA), and serum amyloid A (latex agglutination assay). Data were analyzed using three-way and two-way ANOVA. On Day 0, the combined challenge of trailer transport and exercise significantly increased plasma iohexol in both feeding groups; this increase was not seen in SED horses. On Day 28, EX increased plasma iohexol only in the CO feeding group; this increase was completely prevented by the provision of SUPP. It is concluded that combined transport and exercise induce gastrointestinal hyperpermeability. Dietary SUPP prevents this and therefore may be a useful prophylactic for pathologies associated with gastrointestinal hyperpermeability in horses.

Effects of Aspergillus oryzae prebiotic on dietary energy and nutrient digestibility of growing pigs.

The objective of this study was to determine the effects of Aspergillus oryzae prebiotic (AOP) on nutrient digestibility in growing pigs fed high-fiber diets. Eighteen growing barrows (initial body weight = 50.6 ± 4.9 kg) were surgically equipped with a T-cannula at the distal ileum. Corn and soybean meal-based diets were formulated with fiber from cereal grain byproducts corn (distillers dried grains with solubles, DDGS), rice (rice bran, RB), or wheat (wheat middlings, WM) to meet or exceed all nutrient requirements for 50 to 75 kg growing pigs. Three additional diets were formulated to contain 0.05% AOP supplemented at the expense of corn in the DDGS diet (DDGS + AOP), RB diet (RB + AOP), and WM diet (WM + AOP). All diets contained 0.5% of titanium dioxide as an indigestible marker. Pigs were allotted randomly to a triplicated 6 × 2 Youden square design with six diets and two successive periods. Ileal digesta and fecal samples were collected for 2 d after a 21-d adaptation period, and dry matter (DM), gross energy (GE), crude protein (CP), ether extract (EE), neutral detergent fiber (NDF), and ash were analyzed to calculate apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD). Standardized ileal digestibility (SID) of amino acids (AA) was calculated by correcting AID with basal endogenous AA losses from the same set of pigs. Pigs fed the DDGS+AOP diet had greater (P < 0.05) AID of EE compared with those fed the DDGS diet. However, supplementation of AOP did not (P > 0.05) affect AID of GE, DM, CP, NDF, ash or SID of AA of any high-fiber diet. Supplementation of 0.05% AOP increased (P < 0.05) ATTD of DM, GE, CP, NDF, and ash in DDGS, RB, and WM diets. Diet digestible energy was 35 kcal/kg greater (P < 0.05) in pigs fed AOP supplemented diets compared with those fed diets without AOP. In conclusion, supplementation of AOP increased ATTD of nutrients and energy value in high-fiber diets containing DDGS, RB, or WM.

Exploring genetic variation and population structure in a threatened species, Noturus placidus, with whole-genome sequence data.

The Neosho madtom (Noturus placidus) is a small catfish, generally less than 3 inches in length, unique to the Neosho-Spring River system within the Arkansas River Basin. It was federally listed as threatened in 1990, largely due to habitat loss. For conservation efforts, we generated whole-genome sequence data from 10 Neosho madtom individuals originating from 3 geographically separated populations to evaluate genetic diversity and population structure. A Neosho madtom genome was de novo assembled, and genome size and content were assessed. Single nucleotide polymorphisms were assessed from de Bruijn graphs, and via reference alignment with both the channel catfish (Ictalurus punctatus) reference genome and Neosho madtom reference genome. Principal component analysis and structure analysis indicated weak population structure, suggesting fish from the 3 locations represent a single population. Using a novel method, genome-wide conservation and divergence between the Neosho madtom, channel catfish, and zebrafish (Danio rerio) was assessed by pairwise contig alignment, which demonstrated that genes important to embryonic development frequently had conserved sequences. This research in a threatened species with no previously published genomic resources provides novel genetic information to guide current and future conservation efforts and demonstrates that using whole-genome sequencing provides detailed information of population structure and demography using only a limited number of rare and valuable samples.

Identification of bovine CpG SNPs as potential targets for epigenetic regulation via DNA methylation.

Methylation patterns established and maintained at CpG sites may be altered by single nucleotide polymorphisms (SNPs) within these sites and may affect the regulation of nearby genes. Our aims were to: 1) identify and generate a database of SNPs potentially subject to epigenetic control by DNA methylation via their involvement in creating, removing or displacing CpG sites (meSNPs), and; 2) investigate the association of these meSNPs with CpG islands (CGIs), and with methylation profiles of DNA extracted from tissues from cattle with divergent feed efficiencies detected using MIRA-Seq. Using the variant annotation for 56,969,697 SNPs identified in Run5 of the 1000 Bull Genomes Project and the UMD3.1.1 bovine reference genome sequence assembly, we identified and classified 12,836,763 meSNPs according to the nature of variation created at CpGs. The majority of the meSNPs were located in intergenic regions (68%) or introns (26.3%). We found an enrichment (p<0.01) of meSNPs located in CGIs relative to the genome as a whole, and also in differentially methylated sequences in tissues from animals divergent for feed efficiency. Seven meSNPs, located in differentially methylated regions, were fixed for methylation site creating (MSC) or destroying (MSD) alleles in the differentially methylated genomic sequences of animals differing in feed efficiency. These meSNPs may be mechanistically responsible for creating or deleting methylation targets responsible for the differential expression of genes underlying differences in feed efficiency. Our methyl SNP database (dbmeSNP) is useful for identifying potentially functional "epigenetic polymorphisms" underlying variation in bovine phenotypes.

Genetic diversity and population structure of South African smallholder farmer sheep breeds determined using the OvineSNP50 beadchip.

A population structure study was performed in South African ovine populations using the OvineSNP50 beadchip. Blood samples were obtained from 295 sheep of which 172 had been identified as smallholder Dorpers, 4 smallholder White Dorpers, 46 purebred Dorpers, 26 purebred South African Mutton Merinos and 47 purebred Namaqua Afrikaners. Blood from the latter three breeds were obtained from a resource flock maintained on the Nortier research farm. Genetic diversity was estimated using allelic richness (A r), observed heterozygosity (H o), expected heterozygosity (H e) and inbreeding coefficient (F). Population structure analysis was performed using fastSTRUCTURE to determine the breed composition of each genotyped individual. The Namaqua Afrikaner had the lowest H e of 0.280 ± 0.18 while the H e of smallholder Dorper, Dorper and South African Mutton Merino did not differ and were 0.364 ± 0.13, 0.332 ± 0.16 and 0.329 ± 0.17, respectively. The average inbreeding coefficient was highest for the pure breeds, Namaqua Afrikaner, Dorper and South African Mutton Merino compared to the average inbreeding coefficient for the smallholder Dorper population. The smallholder Dorper were introgressed with Namaqua Afrikaner, South African Mutton Merino and White Dorpers. Similarly, the smallholder Dorper population was more genetically diverse than the purebred Dorper, South African Mutton Merino and Namaqua Afrikaner from the research farm. The higher genetic diversity among the smallholder sheep may be advantageous for their fitness and can be used to facilitate selective breeding.

Elucidating the genetic basis of an oligogenic birth defect using whole genome sequence data in a non-model organism, Bubalus bubalis.

Recent strong selection for dairy traits in water buffalo has been associated with higher levels of inbreeding, leading to an increase in the prevalence of genetic diseases such as transverse hemimelia (TH), a congenital developmental abnormality characterized by absence of a variable distal portion of the hindlimbs. Limited genomic resources available for water buffalo required an original approach to identify genetic variants associated with the disease. The genomes of 4 bilateral and 7 unilateral affected cases and 14 controls were sequenced. A concordance analysis of SNPs and INDELs requiring homozygosity unique to all unilateral and bilateral cases revealed two genes, WNT7A and SMARCA4, known to play a role in embryonic hindlimb development. Additionally, SNP alleles in NOTCH1 and RARB were homozygous exclusively in the bilateral cases, suggesting an oligogenic mode of inheritance. Homozygosity mapping by whole genome de novo assembly also supported oligogenic inheritance; implicating 13 genes involved in hindlimb development in bilateral cases and 11 in unilateral cases. A genome-wide association study (GWAS) predicted additional modifier genes. Although our data show a complex inheritance of TH, we predict that homozygous variants in WNT7A and SMARCA4 are necessary for expression of TH and selection against these variants should eradicate TH.

Lessons for livestock genomics from genome and transcriptome sequencing in cattle and other mammals.

BACKGROUND: Decreasing sequencing costs and development of new protocols for characterizing global methylation, gene expression patterns and regulatory regions have stimulated the generation of large livestock datasets. Here, we discuss experiences in the analysis of whole-genome and transcriptome sequence data. METHODS: We analyzed whole-genome sequence (WGS) data from 132 individuals from five canid species (Canis familiaris, C. latrans, C. dingo, C. aureus and C. lupus) and 61 breeds, three bison (Bison bison), 64 water buffalo (Bubalus bubalis) and 297 bovines from 17 breeds. By individual, data vary in extent of reference genome depth of coverage from 4.9X to 64.0X. We have also analyzed RNA-seq data for 580 samples representing 159 Bos taurus and Rattus norvegicus animals and 98 tissues. By aligning reads to a reference assembly and calling variants, we assessed effects of average depth of coverage on the actual coverage and on the number of called variants. We examined the identity of unmapped reads by assembling them and querying produced contigs against the non-redundant nucleic acids database. By imputing high-density single nucleotide polymorphism data on 4010 US registered Angus animals to WGS using Run4 of the 1000 Bull Genomes Project and assessing the accuracy of imputation, we identified misassembled reference sequence regions. RESULTS: We estimate that a 24X depth of coverage is required to achieve 99.5 % coverage of the reference assembly and identify 95 % of the variants within an individual's genome. Genomes sequenced to low average coverage (e.g., <10X) may fail to cover 10 % of the reference genome and identify <75 % of variants. About 10 % of genomic DNA or transcriptome sequence reads fail to align to the reference assembly. These reads include loci missing from the reference assembly and misassembled genes and interesting symbionts, commensal and pathogenic organisms. CONCLUSIONS: Assembly errors and a lack of annotation of functional elements significantly limit the utility of the current draft livestock reference assemblies. The Functional Annotation of Animal Genomes initiative seeks to annotate functional elements, while a 70X Pac-Bio assembly for cow is underway and may result in a significantly improved reference assembly.

What's in your next-generation sequence data? An exploration of unmapped DNA and RNA sequence reads from the bovine reference individual.

BACKGROUND: Next-generation sequencing projects commonly commence by aligning reads to a reference genome assembly. While improvements in alignment algorithms and computational hardware have greatly enhanced the efficiency and accuracy of alignments, a significant percentage of reads often remain unmapped. RESULTS: We generated de novo assemblies of unmapped reads from the DNA and RNA sequencing of the Bos taurus reference individual and identified the closest matching sequence to each contig by alignment to the NCBI non-redundant nucleotide database using BLAST. As expected, many of these contigs represent vertebrate sequence that is absent, incomplete, or misassembled in the UMD3.1 reference assembly. However, numerous additional contigs represent invertebrate species. Most prominent were several species of Spirurid nematodes and a blood-borne parasite, Babesia bigemina. These species are either not present in the US or are not known to infect taurine cattle and the reference animal appears to have been host to unsequenced sister species. CONCLUSIONS: We demonstrate the importance of exploring unmapped reads to ascertain sequences that are either absent or misassembled in the reference assembly and for detecting sequences indicative of parasitic or commensal organisms.