Mineral Phosphorus Supply in Piglets Impacts the Microbial Composition and Phytate Utilization in the Large Intestine.

A sufficient supply of phosphorus (P) to pigs in livestock farming is based on the optimal use of plant-based phytate and mineral P supplements to ensure proper growth processes and bone stability. However, a high P supplementation might bear the risk of higher environmental burden due to the occurrence of excess P and phytate degradation products in manure. In this context, the intestinal microbiota is of central importance to increase P solubility, to employ non-mineral P by the enzymatic degradation of phytate, and to metabolize residual P. A feeding experiment was conducted in which piglets were fed diets with different P levels, resulting in three groups with low, medium (covering requirements), and high concentrations of available P. Samples from caecum and colon digesta were analysed for microbial composition and phytate breakdown to estimate the microbial contribution to metabolize P sources. In terms of identified operational taxonomic units (OTU), caecum and colon digesta under the three feeding schemes mainly overlap in their core microbiome. Nevertheless, different microbial families correlate with increased dietary P supply. Specifically, microbes of Desulfovibrionaceae, Pasteurellaceae, Anaerovoracaceae, and Methanobacteriaceae were found significantly differentially abundant in the large intestine across the dietary treatments. Moreover, members of the families Veillonellaceae, Selenomonadaceae, and Succinivibrionaceae might contribute to the observed phytate degradation in animals fed a low P diet. In this sense, the targeted manipulation of the intestinal microbiota by feeding measures offers possibilities for the optimization of intestinal phytate and P utilization.

Transcriptional responses in jejunum of two layer chicken strains following variations in dietary calcium and phosphorus levels.

BACKGROUND: Calcium (Ca) and phosphorus (P) are essential nutrients that are linked to a large array of biological processes. Disturbances in Ca and P homeostasis in chickens are associated with a decline in growth and egg laying performance and environmental burden due to excessive P excretion rates. Improved utilization of minerals in particular of P sources contributes to healthy growth while preserving the finite resource of mineral P and mitigating environmental pollution. In the current study, high performance Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB) hens at peak laying performance were examined to approximate the consequences of variable dietary Ca and P supply. The experimental design comprised four dietary groups with standard or reduced levels of either Ca or P or both (n = 10 birds per treatment group and strain) in order to stimulate intrinsic mechanisms to maintain homeostasis. Jejunal transcriptome profiles and the systemic endocrine regulation of mineral homeostasis were assessed (n = 80). RESULTS: Endogenous mechanisms to maintain mineral homeostasis in response to variations in the supply of Ca and P were effective in both laying hen strains. However, the LSL and LB appeared to adopt different molecular pathways, as shown by circulating vitamin D levels and strain-specific transcriptome patterns. Responses in LSL indicated altered proliferation rates of intestinal cells as well as adaptive responses at the level of paracellular transport and immunocompetence. Endogenous mechanisms in LB appeared to involve a restructuring of the epithelium, which may allow adaptation of absorption capacity via improved micro-anatomical characteristics. CONCLUSIONS: The results suggest that LSL and LB hens may exhibit different Ca, P, and vitamin D requirements, which have so far been neglected in the supply recommendations. There is a demand for trial data showing the mechanisms of endogenous factors of Ca and P homeostasis, such as vitamin D, at local and systemic levels in laying hens.

Effects of excessive or restricted phosphorus and calcium intake during early life on markers of bone architecture and composition in pigs.

Sufficient supply of pigs with calcium (Ca) and phosphorus (P) is essential for animal health and welfare during the growth period. However, the P content in animal manure is considered as a cause of massive environmental problems in soil and aquatic ecosystems. To complement previous findings, the objective of this study is the investigation of effects of a reduced and increased Ca and P supplementation on bone mineralization and bone structure compared with the current dietary recommendation. Another aim is to find possible serum markers that would allow the assessment of adequacy of P supply for bone health during growth. The result validated that the recommended Ca and P supply is sufficient, without the addition of microbial phytases. However, addition of P has no further beneficial effects on bone stability, while P supplementation below the recommended level affects bone development and growth performance. Reduced P levels have consequences for cancellous bone density and trabecular architecture. Further fine-tuning of the P supply in conjunction with an appropriate Ca supply will contribute to a reduction in P waste and associated environmental impact while maintaining animal health and welfare.

Reduced phosphorus intake throughout gestation and lactation of sows is mitigated by transcriptional adaptations in kidney and intestine.

BACKGROUND: The environmental impact of pig farming need to be reduced, with phosphorus (P) being of particular interest. Specified dietary regimens and management systems contribute to meet environmental concerns and reduce economic constrains. However, pregnant and lactating sows represent vulnerable individuals, whose reproductive potential and metabolic health status relies on adequate supply of macro- and micronutrients. The aim of this study was to investigate, whether sows fed with a dietary P content that is below or above current recommendations are capable to maintain mineral homeostasis during the reproduction cycle and which endogenous mechanisms are retrieved therefore in kidney and jejunum. Nulliparous gilts were fed iso-energetic diets with recommended (M), reduced (L), or high (H) amounts of mineral P supplements throughout gestation and lactation periods. Blood metabolites and hormones referring to the P homeostasis were retrieved prior to term (110 days of gestation) and at weaning (28 days of lactation). Transcriptional responses in kidney cortex and jejunal mucosa were analyzed using RNA sequencing. RESULTS: The variable dietary P content neither led to an aberration on fertility traits such as total weaned piglets nor to an effect on the weight pattern throughout gestation and lactation. Serum parameters revealed a maintained P homeostasis as reflected by unaltered inorganic P and calcium levels in L and H fed groups. The serum calcitriol levels were increased in lactating L sows. The endocrine responses to the dietary challenge were reflected at the transcriptional level. L diets led to an increase in CYP27B1 expression in the kidney compared to the H group and to an altered gene expression associated with lipid metabolism in the kidney and immune response in the jejunum. CONCLUSIONS: Our results suggest that current P requirements for gestating and lactating sows are sufficient and over supplementation of mineral P is not required. Shifts in renal and jejunal expression patterns between L and H groups indicate an affected intermediate metabolism, which long-term relevance needs to be further clarified.

Comfrey (Symphytum spp.) as an alternative field crop contributing to closed agricultural cycles in chicken feeding.

Local cultivars of comfrey (Symphytum spp.) have been used to cover protein and mineral requirements of farm animals in low-input systems. Due to its known health-promoting (e.g. allantoin), but also anti-nutritive ingredients (e.g. pyrrolizidine alkaloids), multidisciplinary approaches are essential in order to quantify the nutritional value and the potential of its use in poultry and farm animals in terms of meeting animal needs, using local resources as well as remediating over-fertilized soils. Focusing on animal effects, here one-day old sexed Cobb500 broiler chickens were subjected to either a standard control diet or a standard diet supplemented with 4% dried comfrey leaves for 32 days. Performance traits indicate good acceptance of supplementation with comfrey leaves. Parameters for liver function, mineral homeostasis, bone mineral density as well as intestinal microanatomy revealed no signs of impairment. Quantified pyrrolizidine alkaloids were below the detection limit in liver and breast muscle (<5 µg/kg tissue). Comfrey supplemented male broiler chickens showed higher ash content in breast muscle and revealed altered gene expression profiles for metabolic pathways in blood cells. In healthy broiler chickens, the transcriptome analyses revealed no aberrations in the immune-related pathways due to comfrey supplementation. The results imply that the use of comfrey leaves in a high-performance broiler line seems feasible and offers the potential for closed nutrient cycles in site-adapted local agricultural systems. Further analyses need to focus on possible growth-promoting and health-improving components of comfrey and the safe use of chicken products for human consumption.

Ileal Transcriptome Profiles of Japanese Quail Divergent in Phosphorus Utilization.

Phosphorus (P) is an essential component for all living beings. Low P diets prompt phenotypic and molecular adaptations to maintain P homeostasis and increase P utilization (PU). Knowledge of the molecular mechanisms of PU is needed to enable targeted approaches to improve PU efficiency and thus lower P excretion in animal husbandry. In a previous population study, Japanese quail were subjected to a low P diet lacking mineral P and exogenous phytase. Individual PU was determined based on total P intake and excretion. A subset of 20 extreme siblings discordant for PU was selected to retrieve gene expression patterns of ileum (n = 10 per PU group). Sequencing reads have been successfully mapped to the current Coturnix japonica reference genome with an average mapping rate of 86%. In total, 640 genes were found to be differentially abundant between the low and high PU groups (false discovery rate ≤ 0.05). Transcriptional patterns suggest a link between improved PU and mitochondrial energy metabolism, accelerated cell proliferation of enterocytes, and gut integrity. In assessing indicators of the efficient use of macro- and micronutrients, further research on turnover and proliferation rates of intestinal cells could provide an approach to improve P efficiency in poultry species.

Identification of the Key Molecular Drivers of Phosphorus Utilization Based on Host miRNA-mRNA and Gut Microbiome Interactions.

Phosphorus is an essential mineral for all living organisms and a limited resource worldwide. Variation and heritability of phosphorus utilization (PU) traits were observed, indicating the general possibility of improvement. Molecular mechanisms of PU, including host and microbial effects, are still poorly understood. The most promising molecules that interact between the microbiome and host are microRNAs. Japanese quail representing extremes for PU were selected from an F2 population for miRNA profiling of the ileal tissue and subsequent association with mRNA and microbial data of the same animals. Sixty-nine differentially expressed miRNAs were found, including 21 novel and 48 known miRNAs. Combining miRNAs and mRNAs based on correlated expression and target prediction revealed enrichment of transcripts in functional pathways involved in phosphate or bone metabolism such as RAN, estrogen receptor and Wnt signaling, and immune pathways. Out of 55 genera of microbiota, seven were found to be differentially abundant between PU groups. The study reveals molecular interactions occurring in the gut of quail which represent extremes for PU including miRNA-16-5p, miR-142b-5p, miR-148a-3p, CTDSP1, SMAD3, IGSF10, Bacteroides, and Alistipes as key indicators due to their trait-dependent differential expression and occurrence as hub-members of the network of molecular drivers of PU.

Transcriptome profiles of hypothalamus and adrenal gland linked to haplotype related to coping behavior in pigs.

The hypothalamic-pituitary-adrenal (HPA) axis is an important component of neuroendocrine stress regulation and coping behavior. Transcriptome profiles of the hypothalamus and adrenal gland were assessed to identify molecular pathways and candidate genes for coping behavior in pigs. Ten each of high- (HR) and low- (LR) reactive pigs (n = 20) were selected for expression profiling based haplotype information of a prominent QTL-region on SSC12 discovered in our previous genome-wide association study (GWAS) on coping behavior. Comparing the HR and LR pigs showed 692 differentially expressed genes (DEGs) in the adrenal gland and 853 DEGs in the hypothalamus, respectively. Interestingly, 47% (17 out of 36) of DEGs found in both tissues were located in GWAS regions identified on SSC12, indicating that there are significant functional positional candidate genes for coping behaviour. Pathway analysis assigned DEGs to glucocorticoid receptor signaling in the adrenal gland. Furthermore, oxidative phosphorylation, mitochondrial dysfunction, and NGF signaling as well as cholecystokinin/Gastrin-mediated were identified in the hypothalamus. We narrowed the list of candidate genes in GWAS regions by analyzing their DEGs in the HPA axis. The top identified transcripts, including ATP1B2, AURKB, MPDU1 and NDEL1 provide evidence for molecular correlates of coping behavior in GWAS regions.

Deep sequencing of small non-coding RNA highlights brain-specific expression patterns and RNA cleavage.

With the advance of high-throughput sequencing technology numerous new regulatory small RNAs have been identified, that broaden the variety of processing mechanisms and functions of non-coding RNA. Here we explore small non-coding RNA (sncRNA) expression in central parts of the physiological stress and anxiety response system. Therefore, we characterize the sncRNA profile of tissue samples from Amygdala, Hippocampus, Hypothalamus and Adrenal Gland, obtained from 20 pigs. Our analysis reveals that all tissues but Amygdala and Hippocampus possess distinct, tissue-specific expression pattern of miRNA that are associated with Hypoxia, stress responses as well as memory and fear conditioning. In particular, we observe marked differences in the expression profile of limbic tissues compared to those associated to the HPA/stress axis, with a surprisingly high aggregation of 3´-tRNA halves in Amygdala and Hippocampus. Since regulation of sncRNA and RNA cleavage plays a pivotal role in the central nervous system, our work provides seminal insights in the role/involvement of sncRNA in the transcriptional and post-transcriptional regulation of negative emotion, stress and coping behaviour in pigs, and mammals in general.

Breed, Diet, and Interaction Effects on Adipose Tissue Transcriptome in Iberian and Duroc Pigs Fed Different Energy Sources.

In this study, we analyzed the effects of breed, diet energy source, and their interaction on adipose tissue transcriptome in growing Iberian and Duroc pigs. The study comprised 29 Iberian and 19 Duroc males, which were kept under identical management conditions except the nutritional treatment. Two isoenergetic diets were used with 6% high oleic sunflower oil (HO) or carbohydrates (CH) as energy sources. All animals were slaughtered after 47 days of treatment at an average live weight of 51.2 kg. Twelve animals from each breed (six fed each diet) were employed for ham subcutaneous adipose tissue RNA-Seq analysis. The data analysis was performed using two different bioinformatic pipelines. We detected 837 and 1456 differentially expressed genes (DEGs) according to breed, depending on the pipeline. Due to the strong effect of breed on transcriptome, the effect of the diet was separately evaluated in the two breeds. We identified 207 and 57 DEGs depending on diet in Iberian and Duroc pigs, respectively. A joint analysis of both effects allowed the detection of some breed-diet interactions on transcriptome, which were inferred from RNA-Seq and quantitative PCR data. The functional analysis showed the enrichment of functions related to growth and tissue development, inflammatory response, immune cell trafficking, and carbohydrate and lipid metabolism, and allowed the identification of potential regulators. The results indicate different effects of diet on adipose tissue gene expression between breeds, affecting relevant biological pathways.

Feed-efficient pigs exhibit molecular patterns allowing a timely circulation of hormones and nutrients.

Feed efficiency (FE) is a measure of the rate between feed intake and body weight gain and is subject to constant progress in pigs, based on extensive performance tests and analyses of physiological parameters. However, endocrine regulatory circuits that comprise the sensation and perception of intrinsic requirements and appropriate systemic responses have not yet been fully elucidated. It is hypothesized that the gut-brain axis, which is a network of hierarchical anterior regulatory tissues, contributes largely to variations in FE. Therefore, full-sib pigs with extreme residual feed intake values were assigned to experimental groups of high and low FE. Relevant hormones, minerals, and metabolites including fatty acid profiles were analyzed in serum to assess postprandial conditions. Transcriptome profiles were deduced from intestinal (duodenum, jejunum, ileum) and neuroendocrine tissues (hypothalamus). Serum analyses of feed-efficient animals showed an increased content of the incretin GIP, calcium, magnesium, ß-hydroxybutyric acid, and fat compared with low-FE pigs. Complementary expression profiles in intestinal tissues indicate a modulated permeability and host-microbe interaction in FE-divergent pigs. Transcriptomic analyses of the hypothalamus showed that differences between the FE groups in appetite and satiety regulation are less pronounced. However, hypothalamic abundance of transcripts like ADCY7, LHCGR, and SLC2A7 and molecular signatures in local and systemic tissue sites indicate that increased allocation and circulation of energy equivalents, minerals, and hormones are promoted in feed-efficient animals. Overall, patterns of gastrointestinal hormones and gene expression profiles identified host-microbiota interaction, intestinal permeability, feed intake regulation, and energy expenditure as potential mechanisms affecting FE in pigs.

Lower dietary phosphorus supply in pigs match both animal welfare aspects and resource efficiency.

Dietary phosphorus frequently exceeds age-specific requirements and pig manure often contains high phosphorus load which causes environmental burden at regional scales. Therefore, feeding strategies towards improved phosphorus efficiency and reduced environmental phosphorus load have to be developed. A 5-week feeding trial was conducted: piglets received medium, lower (-25%), or higher (+25%) amounts of phosphorus and calcium. Dietary responses were reflected by performance parameters, bone characteristics, and molecular data retrieved from serum, intestinal mucosa, and kidney cortex (p < 0.05). Transcripts associated with vitamin D hydroxylation (Cyp24A1, Cyp27A1, Cyp27B1) were regulated by diet at local tissue sites. Low-fed animals showed attempts to maintain mineral homoeostasis via intrinsic mechanisms, whereas the high-fed animals adapted at the expense of growth and development. Results suggest that a diet containing low phosphorus and calcium levels might be useful to improve resource efficiency and to reduce phosphorus losses along the agricultural value chain.

Sex-Specific Muscular Maturation Responses Following Prenatal Exposure to Methylation-Related Micronutrients in Pigs.

Supplementation of micronutrients involved in DNA methylation, particularly during pregnancy, is recommended because of its impacts on human health, but further evidence is needed regarding the effects of over-supplementation and differences between sexes. Here, a porcine model was used to assess effects of maternal supplementation with one-carbon-cycle compounds during prenatal and postnatal stages on offspring muscle development. Sows received either a standard diet (CON) or a standard diet supplemented with folate, B6, B12, methionine, choline, and zinc (MET) throughout gestation. Myogenesis-, growth-, and nutrient utilization-related transcript expression was assessed using quantitative PCR. Organismal phenotype and gene expression effects differed significantly between males and females. Male MET-offspring showed increased fetal weight during late pregnancy but decreased live weight postnatally, with compensatory transcriptional responses comprising myogenic key drivers (Pax7, MyoD1, myogenin). In contrast, female weights were unaffected by diet, and mRNA abundances corresponded to a phenotype of cellular reorganization via FABP3, FABP4, SPP1 and Insulin-like Growth Factor-signaling. These findings in an animal model suggest that supplementation during pregnancy with methylation-related micronutrients can promote sex-specific myogenic maturation processes related to organismal growth and muscle metabolism. The usage of maternal dietary supplements should be more carefully considered regarding its ability to promote fetal and postnatal health.

Toward improved phosphorus efficiency in monogastrics-interplay of serum, minerals, bone, and immune system after divergent dietary phosphorus supply in swine.

Phosphorus (P) is of vital importance for many aspects of metabolism, including bone mineralization, blood buffering, and energy utilization. In order to identify molecular routes affecting intrinsic P utilization, we address processes covering P intake, uptake, metabolism, and excretion. In particular, the interrelation of bone tissue and immune features is of interest to approximate P intake to animal's physiology and health status. German Landrace piglets received different levels of digestible phosphorus: recommended, higher, or lower amounts. At multiple time points, relevant serum parameters were analyzed and radiologic studies on bone characteristics were performed. Peripheral blood mononuclear cells were collected to assess differential gene expression. Dietary differences were reflected by serum phosphorus, calcium, parathyroid hormone, and vitamin D. Bone reorganization was persistently affected as shown by microstructural parameters, cathepsin K levels, and transcripts associated with bone formation. Moreover, blood expression patterns revealed a link to immune response, highlighting bidirectional loops comprising bone formation and immune features, where the receptor-activator of NF-κB ligand/receptor-activator of NF-κB kinase system may play a prominent role. The modulated P supplementation provoked considerable organismal plasticity. Genes found to be differentially expressed due to variable P supply are involved in pathways relevant to P utilization and are potential candidate genes for improved P efficiency.

Integrated Genome-wide association and hypothalamus eQTL studies indicate a link between the circadian rhythm-related gene PER1 and coping behavior.

Animal personality and coping styles are basic concepts for evaluating animal welfare. Struggling response of piglets in so-called backtests early in life reflects their coping strategy. Behavioral reactions of piglets in backtests have a moderate heritability, but their genetic basis largely remains unknown. Here, latency, duration and frequency of struggling attempts during one-minute backtests were repeatedly recorded of piglets at days 5, 12, 19, and 26. A genome-wide association study for backtest traits revealed 465 significant SNPs (FDR ≤ 0.05) mostly located in QTL (quantitative trait locus) regions on chromosome 3, 5, 12 and 16. In order to capture genes in these regions, 37 transcripts with significant SNPs were selected for expressionQTL analysis in the hypothalamus. Eight genes (ASGR1, CPAMD8, CTC1, FBXO39, IL19, LOC100511790, RAD51B, UBOX5) had cis- and five (RANGRF, PER1, PDZRN3, SH2D4B, LONP2) had trans-expressionQTL. In particular, for PER1, with known physiological implications for maintenance of circadian rhythms, a role in coping behavior was evidenced by confirmed association in an independent population. For CTC1 a cis-expression QTL and the consistent relationship of gene polymorphism, mRNA expression level and backtest traits promoted its link to coping style. GWAS and eQTL analyses uncovered positional and functional gene candidates for coping behavior.