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The natural substitution Ala610Val in the porcine glucocorticoid receptor (GR Ala610Val ) leads to a profound compensatory downregulation of the hypothalamic-pituitary-adrenal (HPA) axis in early ontogeny. In this study, we leveraged this unique animal model to explore mechanisms of HPA axis regulation and consequences of its genetically-based persistent hypoactivity. To this end, we examined transcriptional signature of GR Ala610Val in the hypothalamus, hippocampus, amygdala and adrenal gland in resting conditions (i.e. baseline glucocorticoid level) using mRNA sequencing. In addition, we studied transcriptome responses to two different doses of dexamethasone in the hypothalamus and hippocampus, depending on GR Ala610Val . Across tissues, GR Ala610Val consistently influenced the expression of several clustered protocadherins, particularly PCDHB7. Clustered protocadherins play an important role in neuronal connectivity and are implicated in different neurobiological disorders. Moreover, in line with our previous findings in blood immune cells, we found higher expression of pro-inflammatory genes, including canonical members of the TLR4 signaling pathway, in the brain of Val carriers. While the pro-inflammatory priming occurs already at resting conditions in the amygdala, in hypothalamus and hippocampus this seems to be associated with a stronger downregulation of several marker genes of homeostatic microglia, such as SALL1, by dexamethasone in Val carriers. Regarding the regulation of the HPA axis, GR Ala610Val showed a dose-dependent effect on the central regulator of the axis, CRH, suggesting a dynamic adaptation to the glucocorticoid hypersensitivity of the Val variant. In the adrenal gland, GR Ala610Val appears to downregulate cortisol production by impairing mitochondrial function. Overall, the transcriptional signature of GR Ala610Val provides strong evidence that GR hypersensitivity leads to increased susceptibility to brain disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The objective of this study was to characterize intestinal phytate degradation and mineral utilization by 2 laying hen strains before and after the onset of egg laying using diets without or with a mineral phosphorus (P) supplement. One offspring of 10 roosters per strain (Lohmann Brown-classic [LB] and Lohmann LSL-classic [LSL]) was sacrificed before (wk 19) and after (wk 24) the onset of egg-laying activity and following 4 wk placement in a metabolic unit. Diets were corn-soybean meal-based and without supplemented P (P-) or with 1 g/kg supplemented P (P+) from monocalcium phosphate. In wk 19 and 24, the blood plasma and digesta of duodenum+jejunum and distal ileum were collected. The concentration of P in blood plasma was higher in hens fed P+ than P- (P < 0.001). In duodenum + jejunum and ileum content, the concentrations of InsP 6 , Ins(1,2,4,5,6)P 5 and Ins(1,2,3,4,5)P 5 were lower in P- than in P+ (P ≤ 0.009). In duodenum+jejunum, the concentrations of InsP 6 , Ins(1,2,4,5,6)P 5 and Ins(1,2,3,4,5)P 5 were lower in wk 24 than 19 and lower in LSL than LB hens (P < 0.001). The concentration of myo-inositol (MI) in duodenum + jejunum content was lower in wk 19 than 24 (P < 0.001). Following a 4-d total excreta collection, the retained amount of P was higher in P+ than P- (P < 0.001). Phosphorus retention was lower in LB hens fed P- than in other treatments (P × strain: P = 0.039). In the jejunal tissue, some genes related to intracellular InsP metabolism were higher expressed in LB than LSL hens. The renunciation of mineral P increased endogenous phytate degradation, but more P was retained with supplemented P. Differences in endogenous phytate degradation between the periods before and after the onset of egg laying might be attributed to different Ca concentrations in intestinal digesta caused by different Ca needs in both periods., Competing Interests: DISCLOSURES MFö received speaker fees from Kyowa Kirin without relevance to this study. The authors declare they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Phosphorus (P) is an essential mineral for all forms of life including laying hens, playing a crucial role in growth and efficient egg production. Recent studies suggest that current P recommendations might exceed the physiological demand, leading to unnecessarily high P excretions. This study on Lohmann Brown (LB) and Lohmann Selected Leghorn (LSL) laying hens (n=80; 10 replicates per strain, production period, and dietary group) investigates transcriptional changes in the jejunum, a critical intestinal segment for mineral absorption, in response to a diet either without (P-) or with (P+) a mineral supplement from monocalcium phosphate, administered over a 4-week period during the transition (15-19 weeks) or onset of laying (20-24 weeks). DESeq2 analysis of RNA sequencing data revealed that most differentially expressed genes (DEGs) varied between strains and age groups, with less pronounced effects from dietary mineral P content. The 19-week-old LB hens showed a stronger response to dietary mineral P removal, with transcripts affiliated with increased adaptation of the metabolism and decreased immune pathway activation. The identified pathways such as folate biosynthesis and p53 signaling, potentially link altered energy and amino acid metabolism (2-oxocarboxylic acid and arginine). Interestingly, genes involved in calcium transport (CALB1) and cellular signaling (PRKCA, STEAP4) along with tight junctions (CLDN2) were affected by complete removal of mineral P supplements, suggesting a promoted intestinal mineral uptake. Transcriptional regulation in the jejunum in response to low dietary mineral content is strain-specific when the laying phase begins, which may contribute to a physiological Ca:P ratio., Competing Interests: Disclosures All authors declare no conflicts of interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The objectives of this study were to investigate whether an increased dietary myo-inositol (MI) supply translates into changes in MI concentrations and endogenous mucosal phosphatase activities in the intestine of laying hens and whether different laying hen strains respond differently to MI supplementation. The diets were corn-soybean meal-based and supplemented without (MI0) or with 1 (MI1), 2 (MI2), or 3 (MI3) g MI/kg feed. Ten hens per strain (Lohmann Brown-classic (LB) and Lohmann LSL-classic (LSL)) and diet were sacrificed at the age of 30 wk following a 4-wk stay in a metabolic unit. The blood plasma, digesta of the duodenum+jejunum and distal ileum, mucosa of the duodenum, and eggs were collected at wk 30. The concentration of MI in the blood plasma was increased by MI supplementation (P < 0.001); however, that of MI3 did not further increase compared with MI2. The concentration of MI in the duodenum+jejunum and ileum increased steadily (P < 0.001). The MI concentration in the duodenum+jejunum was higher in LB than in LSL hens (P = 0.017). The MI concentration in egg yolk was increased by MI supplementation (P < 0.001) and was higher in LB than in LSL hens (P = 0.015). Strain or diet did not affect mucosal phosphatase activity. Myo-inositol flow at the terminal ileum and postileal disappearance increased with each increment in MI supplementation (P < 0.001) and was higher in LB than in LSL hens (P ≤ 0.041). Regression analysis indicated that, on average, 84% of supplemented MI was retained in the body or metabolized and excreted in a different form. Based on the measured MI concentrations in the blood and eggs, dietary MI was not completely absorbed in the small intestine and, to a different extent, in the two laying hen strains. A higher dietary MI supply was followed by higher intestinal absorption or metabolism by microorganisms. The fate of supplemented MI and its relevance to birds warrant further research., Competing Interests: Declaration of competing interest MFö received speaker fees unrelated to this study from Kyowa Kirin. The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Efforts to achieve sustainable phosphorus (P) inputs in broiler farming which meet the physiological demand of animals include nutritional intervention strategies that have the potential to modulate and utilize endogenous and microbiota-associated capacities. A temporal P conditioning strategy in broiler nutrition is promising as it induces endocrinal and transcriptional responses to maintain mineral homeostasis. In this context, the current study aims to evaluate the composition of the jejunal microbiota as a functional entity located at the main absorption site involved in nutrient metabolism. Starting from a medium or high P supply in the first weeks of life of broilers, a depletion strategy was applied at growth intervals from d 17 to 24 and d 25 to 37 to investigate the consequences on the composition of the jejunal microbiota. The results on fecal mineral P, calcium (Ca), and phytate contents showed that the diets applied to the depleted and non-depleted cohorts were effective. Microbial diversity in jejunum was represented by alpha diversity indices which appeared unaffected between dietary groups. However, chickens assigned to the dietary P depletion groups showed significantly higher abundances of Facklamia, Lachnospiraceae, and Ruminococcaceae compared to non-depleted control groups. Based on current knowledge of microbial function, these microorganisms make only a minor contribution to the birds' adaptive mechanism in the jejunum following P depletion. Microbial taxa such as Brevibacterium, Brachybacterium, and genera of the Staphylococcaceae family proliferated in a P-enriched environment and might be considered biomarkers for excessive P supply in commercial broiler chickens., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Phosphorus (P) inclusion in broiler diets needs to meet the physiological demands at a specific developmental stage to ensure the performance, health, and welfare of the birds and minimize nutrient losses. Toward a more efficient utilization of P in broiler husbandry, a timed nutritional conditioning strategy might enhance the endogenous mechanisms of mineral homeostasis and thus reduce dietary P supply of mineral sources. In this study, following a variable P supply in the starter phase, the effects of a dietary P depletion of broiler chickens were investigated at different developmental stages. Physiological adaptation mechanisms were elucidated based on zootechnical performance, endocrine parameters, regulation of intestinal P transport, bone characteristics, and health aspects. The results revealed a marked response to P depletion at the earliest developmental phase, after which indications of effective compensatory mechanism were detectable with advancing ages. Potential mechanisms that enable broilers to maintain mineral homeostasis primarily include endocrine control mediated by calcitriol actions, as well as intestinal P uptake and mineral mobilization from the bone. Conclusively, the precise timing, duration, and extent of a P depletion strategy in the broiler chicken might be considered for optimized nutrient utilization., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Aggregation of data, including deep sequencing of mRNA and miRNA data in jejunum mucosa, abundance of immune cells, metabolites, or hormones in blood, composition of microbiota in digesta and duodenal mucosa, and production traits collected along the lifespan, provides a comprehensive picture of lifelong adaptation processes. Here, respective data from two laying hen strains (Lohmann Brown-Classic (LB) and Lohmann LSL-Classic (LSL) collected at 10, 16, 24, 30, and 60 wk of age were analyzed. Data integration revealed strain- and stage-specific biosignatures, including elements indicative of molecular pathways discriminating the strains. Although the strains performed the same, they differed in the activity of immunological and metabolic functions and pathways and showed specific gut-microbiota-interactions in different production periods. The study shows that both strains employ different strategies to acquire and maintain their capabilities under high performance conditions, especially during the transition phase. Furthermore, the study demonstrates the capacity of such integrative analyses to elucidate molecular pathways that reflect functional biodiversity. The bioinformatic reduction of the multidimensional data provides good guidance for further manual review of the data., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Daily and seasonal temperature fluctuations are inevitable due to climate change, which highlights the importance of studying the detrimental effects of temperature fluctuations on the health, productivity, and product quality of farm animals. Muscle membrane composition and the molecular signals are vital for muscle cell differentiation and muscle growth, but their response to temperature stress is not well characterized. Temperature changes can lead to modification of membrane components of the cell, which may affect its surroundings and intracellular signaling pathways. Using C2C12 myoblast cells as a model of skeletal muscle development, this study was designed to investigate the effects of high temperature (39 °C and 41 °C) and low temperature (35 °C) on molecular pathways in the cells as well as the cell membrane fatty acid composition. Our results show that several genes were differentially expressed in C2C12 cells cultured under heat or cold stress, and these genes were enriched important KEGG pathways including PI3K-Akt signaling pathway, lysosome and HIF- signaling pathway, Wnt signaling pathway and AMPK signaling pathway. Our analysis further reveals that several membrane transporters and genes involved in lipid metabolism and fatty acid elongation were also differentially expressed in C2C12 cells cultured under high or low temperature. Additionally, temperature stress shifts the fatty acid composition in the cell membranes, including the proportion of saturated, monounsaturated and polyunsaturated fatty acids. This study revealed an interference between fatty acid composition in the membranes and changing molecular pathways including lipid metabolism and fatty acids elongation mediated under thermal stress. These findings will reinforce a better understanding of the adaptive mechanisms in skeletal muscle under temperature stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In smallholder agriculture, the fast-growing and perennial accumulator plant comfrey (Symphytum spp.) was used to supply pigs with protein and minerals. Comfrey leaves show similar values in dry matter as soybean or blue lupine in crude protein content, but much higher levels of calcium and phosphorus. However, in terms of increased efficiency in animal husbandry, comfrey has been displaced by mainly soybean and cereals. Due to its profile of macro- and micronutrients the use of comfrey could have the potential to re-establish local resource cycles and help remediate over-fertilized soils. The aim of the study was to evaluate whether a modern pig breed accepts a continuous feed supplement of dried comfrey leaves. After an initial adaptation period post-weaning, German Landrace piglets were subjected to either a standard control diet or a diet supplemented with 15% dried comfrey leaves for 4 weeks. Body weight was reduced in comfrey-supplemented piglets compared to controls, which might be attributed to reduced palatability in the experimental setting. Nevertheless, comfrey-supplemented piglets exhibited adequate bone mineralization and intestinal integrity. The microbiome profile in feces and digesta revealed higher diversity in comfrey-supplemented piglets compared to controls, with pronounced effects on the abundances of Treponema and Prevotella. This may be due to described bio-positive components of the comfrey plant, as data suggest that the use of comfrey leaves may promote intestinal health. Digestive tract phosphorus levels were reduced in piglets receiving comfrey supplementation, which may ultimately affect phosphorus levels in manure. Results indicate that comfrey leaves could serve as a feed component in integrated agricultural systems to establish regional nutrient cycles. The trial provides a basis for further work on comfrey as a regionally grown protein source and effective replacement for rock mineral supplements., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Urine and fecal excretions from cattle contribute to global nitrogen (N) emissions. The milk urea nitrogen (MUN) concentration in dairy cows is positively correlated with urinary urea N (UUN) emissions, and both decline with the reduction in crude protein intake. However, MUN concentration may differ between individual cows despite feeding the same ration. Thus, we hypothesized that due to differences in endogenous N utilization cows with high MUN concentration excrete more UUN than cows with a low MUN concentration. The objective of the present study was to elucidate N partitioning and urea metabolism in dairy cows with divergent MUN concentrations fed two planes of crude protein. Twenty Holstein dairy cows with high (HMU; n = 10) and low (LMU; n = 10) milk urea concentrations were fed two isocaloric diets with a low (LP) and normal (NP) crude protein level. Methane and ammonia emissions were recorded in respiration chambers. Feed intake, feces and urine excretions and milk yield were recorded for four days and subsamples were analyzed for total N and N-metabolites. A carbon-13 labeled urea bolus was administered intravenously followed by a series of plasma samplings. Total N and UUN excretions and ammonia emissions from excreta were lower on the LP diet, however, methane emissions, urinary N excretions and ammonia emissions were comparable between groups. Although plasma and salivary urea concentrations, urea pool size and urea turnover were higher, HMU cows had lower renal urea clearance rates. Additionally, HMU cows had lower renal clearance rates for creatinine, uric acid and creatine and excreted less uric acid (on the LP diet only) and creatine with urine. In conclusion, contrary to our hypothesis, HMU cows did not excrete more UUN than LMU cows. The lower urinary creatine excretion of HMU cows suggests that these animals have a lower environmental nitrogen footprint., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The objective of this study was to compare 2 laying hen strains in 5 production periods regarding phytase activity, phytate (InsP 6 ) degradation, and myo-inositol (MI) release in the digestive tract and phosphorus (P) and calcium (Ca) utilization. One offspring of 10 nonrelated roosters per strain (Lohmann Brown-classic (LB) or Lohmann LSL-classic (LSL)) was placed in one of 20 metabolic units in a completely randomized block design in week 8, 14, 22, 28, and 58 of life. All hens were fed the same corn and soybean meal-based diet at one time, but the diet composition was adjusted to the requirements in the respective period. For 4 consecutive days, excreta were collected quantitatively at 24-hour intervals. In week 10, 16, 24, 30, and 60, the blood plasma, digesta of crop, gizzard, jejunum, ileum, and ceca, and mucosa of the jejunum was collected. The concentration of inorganic P in the blood plasma was higher in LB than in LSL hens (P = 0.026). Plasma Ca concentrations increased with each period (P < 0.001) in both strains. In jejunum digesta, the MI concentration did not differ between strains, but InsP 6 concentration was higher in LB than in LSL hens (P = 0.002) and the highest in week 30 and 60. Total phosphatase and phytase activities were higher in LB than in LSL hens (P ≤ 0.009). Period effects were also significant for these enzymes. Concentrations of some constituents of the cecal content were different between the strains. The MI concentration in the egg albumen and yolk was higher in LB than in LSL hens. Differences in InsP 6 - and MI-related metabolism of the 2 hen strains existed. These differences were partly dependent of the period. Especially, week 24 was a period of remarkable change of metabolism. Great differences also existed among individuals, making it worth to have a closer look at the metabolism of individuals in addition to evaluating treatment means. Further studies on metabolic, genetic, and microbiome level may help explain these differences., (Copyright © 2020. Published by Elsevier Inc.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Despite the crucial role of glucocorticoid receptor (GR) in proper immune responses, the effect of GR hypersensitivity on inflammation is rarely reported. To fill this knowledge gap, we exploited the natural gain-of-function substitution in the porcine glucocorticoid receptor (GR Ala610Val ) and challenged pigs carrying normal or hypersensitive GR using 50 µg/kg lipopolysaccharide (LPS) following pretreatment with either saline or single bolus of 60 µg/kg dexamethasone (DEX). The GR Ala610Val substitution reduced baseline cortisol, adrenocorticotropic hormone (ACTH), and triglyceride concentration and granulocyte proportion whereas baseline platelet counts were elevated. Val-carriers, i.e. AlaVal as well as ValVal pigs, showed less LPS-induced cortisol rise but the cortisol fold change was similar in all genotypes. Differently, ACTH response to LPS was most significant in GR Ala610Val heterozygotes (AlaVal). LPS-induced disorders, including sickness behaviors, anorexia, thrombocytopenia, cytokine production, and metabolic alterations were more intense in Val-carriers. On the other hand, Val-carriers were more sensitive to DEX effect than wild types (AlaAla) during endotoxemia, but not under unchallenged conditions. This is the first report revealing aggravated responses to endotoxemia by GR gain-of-function. Together, these results imply that GR hypersensitivity is difficult to diagnose but may represent a risk factor for endotoxemia and sepsis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Broiler eggs were either incubated at 37.8°C during the whole incubation period (control), or at higher (38.8°C, group H) and lower temperatures (36.8°C, group L) from embryonic day (ED) 7 up to ED 10 (ED 7 to 10) or from ED 10 up to ED 13 (ED 10 to 13). Before and after this temperature treatment the eggs were incubated at 37.8°C. The day-old chicks were weighted, sexed and fed up to day 35. On days 1 and 35 samples were taken from the breast and leg muscles for analyzing of the mitochondrial respiratory activity (MRA) and from the breast muscles for analysis of the cross-sectional areas (CSA) and the glycogen phosphorylase (GP), phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS) and cytochrome oxidase (COX) activities. Statistical analysis showed that treatment (control, group H, group L), sex and their interaction, but not the treatment period (ED 7 to 10; ED 10 to 13), significantly influenced the results. Group H chicks had lower (P⩽0.05) body and heart weights but higher (P⩽0.05) liver weights, CSA values, leg MRA as well as PFK, LDH, CS, GP and COX activities compared with the group L chicks. The results of the control chicks differ (P⩽0.05) from those of the group H (body, heart weight, COX), the group L chicks (liver weight, PFK, LDH, CS, GP) or the birds of both other groups (CSA). The group H broiler had higher (P⩽0.05) body and leg weights as well as LDH, CS, COX and GP activities than the group L broilers. The BWs and the LDH and GP results of the control broiler differ (P⩽0.05) from those of both other groups or from the results of the group H (CS) and group L broiler (COX). Female broilers had lower (P⩽0.05) body, breast and leg weights, but higher (P⩽0.05) CSA, LDH, CS and GP activities than the male animals. Analysis of treatment×sex interaction showed that group H hens had higher (P⩽0.05) body and breast weights, LDH and GP activities compared with the group L hens, whereas in the male broiler no effect of the interaction could be found, except for the lower (P⩽0.05) CSA values in the group H than group L cocks. The treatment effects are probably due to altered embryonic activity and related molecular mechanisms. The sex-related differences in the broiler indicate that these alterations already occur in the embryos and chicks, but become significant with the sexual dimorphism after hatch.

Residual feed intake (RFI), the difference between actual feed intake and predicted feed requirements, is suggested to impact various aspects of meat quality. The objective of this study was to investigate the molecular mechanisms underpinning the relationship between RFI and meat quality. Technological, sensory and nutritional analysis as well as transcriptome profiling were carried out in Longissimus thoracis et lumborum muscle of pigs divergent in RFI (n=20). Significant differences in sensory profile and texture suggest a minor impairment of meat quality in more efficient pigs. Low RFI animals had leaner carcasses, greater muscle content and altered fatty acid profiles compared to high RFI animals. Accordingly, differentially expressed genes were enriched in muscle growth and lipid & connective tissue metabolism. Differences in protein synthesis and degradation suggest a greater turnover of low RFI muscle, while divergence in connective tissue adhesion may impact tenderness. Fatty acid oxidation tending towards decrease could possibly contribute to reduced mitochondrial activity in low RFI muscle., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The family of steroid hormones is quite attractive for the approach of phenotype monitoring in farm animals. Therefore, we developed a new protocol for the quantitative analysis of natural steroids in follicular fluid from dairy cows. The corresponding steroid profile, which consists of progesterone, corticosterone, hydrocortisone, testosterone, and androstenedione covering three distinct steroid classes, was determined by LC/MS. Quantification is achieved by use of steroid standards diluted in steroid-free follicular fluid as calibrators. Thus, the new protocol does not require deuterated standards. In order to correct for conditional performance of the analytical system we have used dexamethasone as an internal standard. The method was validated according to EMA guidelines. Within- and between-day variations were below 20% for most parameters assessed. All steroids assessed had lower limits of quantification in the range of 2.1 to 4.4ng/ml. We have established a simple and sensitive analytical system in order to step towards a broader and cost-efficient phenotyping analysis in follicular fluid from dairy cows., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The porcine intestinal epithelium is a primary target for mycotoxin deoxynivalenol (DON) and lipopolysaccharides (LPS). Although epithelial cells are exposed to these toxins mainly from the luminal-chyme compartment an exposure from the blood side resulting from systemic absorption cannot be excluded. Thus, we investigated the effect of DON and LPS, alone or combined, on porcine intestinal epithelial cells IPEC-J2 on a transcriptional, translational and functional level when administered either from apical or basolateral. IPEC-J2 cells were cultured on 12-well inserts in complete medium at 5% CO2 and 39°C and subjected to following treatments: control (CON), 2000 ng/mL DON, 1 μg/mL LPS or DON+LPS for 72 h, either from apical or basolateral. Transepithelial electrical resistance (TEER), protein and IL-8 content were measured and microarray analysis, qRT-PCR (IL-8, zonula occludens-1 ZO-1, β-actin), Western Blot (ZO-1, β-actin) and immunofluorescence (ZO-1) were performed. Data of at least three independent experiments were analysed with ANOVA and Dunnett's post hoc test. Basolateral DON resulted in significantly lower cell counts (p<0.05) with larger cells (p<0.01), whereas apical DON reduced total (p<0.001) and specific protein content (IL-8 content CON vs. DON: 2378 pg/3 mL vs. 991 pg/3 mL; p<0.001). Transcripts of ß-actin and ZO-1 were significantly upregulated in response to DON, irrespective of direction, whereas IL-8 mRNA remained unaffected. However, ZO-1 spatial distribution in the tight junction and its function (TEER) were detrimentally affected by basolateral DON only. In conclusion, direction of DON exposure affected IPEC-J2 differently on a translational and functional level, but was mainly inconsequential on a transcriptional level., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The degradation of myofibrillar proteins during meat maturation affects the water holding capacity (WHC) of meat. Our study sought to identify polymorphisms in UBE3B and ZRANB1, genes encoding proteins involved in ubiquitination, and to evaluate the relationship between genotype, transcript abundance, and WHC of pork. A single SNP of ZRANB1, c.552A>G (p.Ile153Val), and two silent SNPs of UBE3B, c.1921A>T and c.4292C>T, were associated with muscle pH, conductivity, meat colour, or drip loss in German Landrace (GL, n=266) and Pietrain×(Large White×German Landrace) (PiF1, n=316). Further, carriers of the minor alleles at the SNPs tended to have increased transcript abundance. Consistent with the protein degradation promoting and inhibiting effects of UBE3B and ZRANB1, respectively, and the expected impact on WHC, their expressions were positively and negatively associated with WHC. The results implicate that the SNPs in both genes are in linkage with a causal site that affects transcript abundance and WHC., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This study aimed to investigate the genes PPARGC1A (peroxisome proliferator-activated receptor gamma-coactivator 1A) and CAPNS1 (calpain small subunit 1) as candidate genes affecting meat quality traits in pigs. Four polymorphisms were identified in PPARCG1A and three in CAPNS1. The PPARGC1A polymorphism c.1288T>A was associated with pH and cooking loss in a F2 Duroc×Pietrain experimental cross (DuPi, n=313) and with pH values in Italian Large White (ILW, n=380) and Italian Landrace (ILA, n=158) populations (P<0.05). The CAPNS1 polymorphism c.429A>C was associated with pH and conductivity in DuPi and with meat color in ILA (P<0.05). PPARGC1A mRNA expression associated with drip loss (P<0.01) and the same tendency was found for CAPNS1 (P=0.06). The promoter methylation profiling suggested that methylation is not involved in CAPNS1 expression regulation. In conclusion, porcine PPARGC1A and CAPNS1 genes may affect meat quality traits, with breed-specific differences, and they could be used as markers for the improvement of meat quality in pigs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The research aimed to screen for polymorphism, expression of Tenascin C (TNC) and association with meat and carcass quality traits. Three single nucleotide polymorphisms were detected. In a Duroc×Pietrain F2 cross (DuPi) population, g.44488C>T was associated with meat color and ham weight; g.68794A>G was associated with pH at 24h post mortem in ham (pH24(H)) and muscle area but g.68841C>T was not statistically associated. Genotyping in a commercial Pietrain (Pi) population showed that g.44488C>T was associated with pH24(H), whereas g.68794A>G was associated with conductivity at 45 min post mortem in loin and backfat thickness. Diplotypes showed significant effects on pH24(H) in both populations. The expression was associated with pH at 45 min post mortem in loin and cooking loss. TNC was significantly higher in animals with higher muscle pH. Linkage analysis revealed four trans-regulated eQTL on four autosomes. These results suggest that TNC could be a potential candidate gene for meat quality traits in pigs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The aim of this research was to screen for polymorphism and to perform an association study of IFI6 with meat and carcass quality traits. A SNP (g.370A>G) was detected which was associated (P<0.05) with meat colour, pH 24h post mortem (p.m.) in ham, conductivity 45 min p.m. in loin and conductivity 24 h p.m. in ham, drip loss and carcass length in Duroc x Pietrain and with meat colour, muscle area and ham percentage in the Pietrain population. Highest expression of IFI6 mRNA was detected in skeletal muscle (longissimus dorsi) by qRT-PCR comparing different tissues. Both qRT-PCR and western blot revealed that the IFI6 gene and protein expressions were significantly (P<0.05) higher in skeletal muscle with low drip loss compared to that of high drip loss. IFI6 protein was localized in the myocytes membrane. Results suggested that IFI6 might play roles in meat and carcass quality and is a potential positional, physiological and functional candidate gene for improving meat quality traits in pigs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Skeletal muscle development in vertebrates - also termed myogenesis - is a highly integrated process. Evidence to date indicates that the processes are very similar across mammals, poultry and fish, although the timings of the various steps differ considerably. Myogenesis is regulated by the myogenic regulatory factors and consists of two to three distinct phases when different fibre populations appear. The critical times when myogenesis is prone to hormonal or environmental influences depend largely on the developmental stage. One of the main mechanisms for both genetic and environmental effects on muscle fibre development is via the direct action of the growth hormone-insulin-like growth factor (GH-IGF) axis. In mammals and poultry, postnatal growth and function of muscles relate mainly to the hypertrophy of the fibres formed during myogenesis and to their fibre-type composition in terms of metabolic and contractile properties, whereas in fish hyperplasia still plays a major role. Candidate genes that are important in skeletal muscle development, for instance, encode for IGFs and IGF-binding proteins, myosin heavy chain isoforms, troponin T, myosin light chain and others have been identified. In mammals, nutritional supply in utero affects myogenesis and the GH-IGF axis may have an indirect action through the partitioning of nutrients towards the gravid uterus. Impaired myogenesis resulting in low skeletal myofibre numbers is considered one of the main reasons for negative long-term consequences of intrauterine growth retardation. Severe undernutrition in utero due to natural variation in litter or twin-bearing species or insufficient maternal nutrient supply may impair myogenesis and adversely affect carcass quality later in terms of reduced lean and increased fat deposition in the progeny. On the other hand, increases in maternal feed intake above standard requirement seem to have no beneficial effects on the growth of the progeny with myogenesis not or only slightly affected. Initial studies on low and high maternal protein feeding are published. Although there are only a few studies, first results also reveal an influence of nutrition on skeletal muscle development in fish and poultry. Finally, environmental temperature has been identified as a critical factor for growth and development of skeletal muscle in both fish and poultry.

Selective breeding is an effective tool to improve livestock. Several selection experiments have been conducted to study direct selection responses as well as correlated responses in traits of skeletal muscle growth and function. Moreover, comparisons of domestic with wild-type species and of extreme breeds provide information on the genetic background of the skeletal muscle phenotype. Structural muscular components that differed with increasing distance in lean growth or meat quality in mammals were found to be myofibre number, myofibre size, proportions of fibre types as well as the numbers and proportions of secondary and primary fibres. Furthermore, markers of satellite cell proliferation, metabolic enzyme activities, glycogen and fat contents, the expression of myosin heavy chain isoforms, of activated AMPKα and other proteins in skeletal muscle tissue and circulating IGF1 and IGF-binding proteins have been identified to be involved in selection responses observed in pigs, cattle and/or chicken. The use of molecular methods for selective breeding of fish has only recently been adopted in aquaculture and studies of the genetic basis of growth and flesh quality traits are scarce. Some of the molecular markers of muscle structure/metabolism in livestock have also been identified in fish, but so far no studies have linked them with selection response. Genome scans have been applied to identify genomic regions exhibiting quantitative trait loci that control traits of interest, for example, muscle structure and meat quality in pigs and growth rate in chicken. As another approach, polymorphisms in candidate genes reveal the relationship between genetic variation and target traits. Thus, in large-scale studies with pigs' associations of polymorphisms in the HMGA2, CA3, EPOR, NME1 and TTN genes with traits of carcass and meat quality were detected. Other studies revealed the significance of mutations in the IGF2 and RYR1 genes for carcass lean and muscle fibre traits in pigs. Mutations in the myostatin (MSTN) gene in fish were also examined. Advances in research of the genetic and environmental control of traits related to meat quality and growth have been made by the application of holistic 'omics' techniques that studied the whole muscle-specific genome, transcriptome and proteome in relation to muscle and meat traits, the development of new methods for muscle fibre typing and the adaptation of biophysical measures to develop parameters of muscle fibre traits as well as the application of in vitro studies. Finally, future research priorities in the field are defined.

Traits related to fatness, important as economic factors in pork production, are associated with serious diseases in humans. Genetical genomics is a useful approach for studying the effects of genetic variation at the molecular level in biological systems. Here we applied a whole-genome association analysis to hepatic gene expression traits, focusing on transcripts with expression levels that correlated with fatness traits in a porcine model. A total of 150 crossbred pigs [Pietrain × (German Large White × German Landrace)] were studied for transcript levels in the liver. The 24K Affymetrix expression microarrays and 60K Illumina single nucleotide polymorphism (SNP) chips were used for genotyping. A total of 663 genes, whose expression significantly correlated with the trait "fat area," were analyzed for enrichment of functional annotation groups as defined in the Ingenuity Pathways Knowledge Base (IPKB). Genes involved in metabolism of various macromolecules and nutrients as well as functions related to dynamic cellular processes correlated with fatness traits. Regions affecting the transcription levels of these genes were mapped and revealed 4,727 expression quantitative trait loci (eQTL) at P < 10⁻⁵, including 448 cis-eQTL. In this study, genome-wide association analysis of trait-correlated expression was successfully used in a porcine model to display molecular networks and list genes relevant to fatness traits.

Pre-slaughter stress has a negative impact on animal welfare and on meat quality. Aggressive behaviour when pigs are mixed together for transportation to, or on arrival at, the abattoir is an important factor in pre-slaughter stress. Aggressiveness of pigs varies between individuals in the population, and this study investigated its effects on stress and meat quality at slaughter. We mixed pigs at a young age to identify individuals of high (H) or low (L) aggressive temperament using the previously validated approach of lesion scoring. To contrast extremes of social stress single-sex groups of eight pigs were mixed according to their aggressiveness in HH, HL or LL combinations or left unmixed (U) prior to transport and slaughter (n = 271). Each treatment was replicated in at least two groups in each of four slaughter batches. Mixing per se had little effect, but mixed groups composed of aggressive pigs (HH) had more carcass skin lesions and higher levels of plasma cortisol at slaughter and had loin muscle samples with higher pH at 24 h, and lower redness (a*) and yellowness (b*) compared to the other treatments. Females had higher levels of plasma cortisol at slaughter, a more rapid decline in pH post-slaughter and greater lean content of meat. Lactate and creatine kinase (CK) levels and meat pH were affected by the interaction of sex and treatment. Genetic factors, dam and sire line composition, and halothane locus (ryanodine receptor 1, RYR1) genotype, also affected a number of production and meat quality parameters as expected. Additionally, 'commercially normal' levels of social stress were studied in four further slaughter batches with no manipulation of group composition (n = 313). In these pigs, the proportion of unfamiliar pigs and group size of lairage groups explained limited variation in lesion scores at slaughter, but earlier aggressiveness did not. High numbers of skin lesions on the carcass were associated with high levels of cortisol and lactate and low glucose at slaughter, but not with meat quality measures. When stress and meat quality measures were compared for all pigs, high lactate was associated with low early pH and high drip loss, while high cortisol and CK were associated with high pH at 24 h and changes in meat colour. In conclusion, mixing pigs of above average aggressiveness resulted in greater aggression and stress, and changes in meat quality parameters, consistent with the effects of pre-slaughter stress on muscle chemistry.

Zyxin (ZYX) is one of the proteins in focal adhesions along the actin fibers playing a role in actin organization and signal transduction. By radiation hybrid and genetic mapping we assigned ZYX to porcine chromosome 18 in the area of quantitative traits loci for carcass and meat quality and muscle fiber traits and hence considered ZYX a functional positional candidate gene. Analysis of a newly detected SNPs (c.+279 C>T, c.+399 A>G, c.+522 A>G) in pigs from different commercial breeds (Pietrain [Pi], German Landrace [LR], German Large White x German Landrace [F1] and PiF1) revealed a significant association with carcass traits (including: side- and backfat thickness, loin weight and carcass lean content) and meat quality traits (including: pH, color and drip loss). However, the lack of consistent association across all pig populations in this study indicates that the association of the SNPs may be depending on causal mutations in linkage disequilibrium and/or interactions with other loci.

Corticotropin-releasing hormone (CRH), a major regulator of neuroendocrine response to stress, is involved in the control of energy balance and thus may affect body composition and growth. The porcine CRH (pCRH) gene was studied as a comparative-positional candidate for QTL for longissimus muscle area, average backfat thickness, carcass length, and average daily gain on test on porcine chromosome 4. Sequence of the complete transcriptional unit of pCRH gene spanning 2068bp was determined along with 582bp of the 5'-flanking region. Cross-species sequence comparison revealed a number of potential regulatory regions including an intronic evolutionary conserved region and an adjacent CpG island that may control cell-type specific expression of the CRH gene. A SNP in exon 2 (c.+83G>A) leading to a non-conservative amino acid exchange (p.28Arg>Gln) in the prohormone was identified that is segregating in the DUMI resource population. Linkage and association analysis based on this SNP revealed that for all four traits the pCRH gene falls in the QTL peak area and that the c.+83G>A SNP shows a highly significant additive effect (p<0.0001). Physical mapping using the IMpRH panel assigned the pCRH gene to interval SW724-S0107, promoting the gene as a positional candidate also for QTL identified in other porcine resource populations. Additional four variable sites were identified that segregate in commercial pig breeds. Particularly interesting is a SNP (g.233C>T) in the 5'-flanking region that occurred in an evolutionary conserved motif. The knowledge of the DNA-variation of pCRH gene will facilitate follow-up studies necessary to provide definite genetic evidence of the effect of pCRH gene on body composition and growth.

It may be possible to avoid inadequate in vitro culture conditions by incubating gametes or embryos in the oviducts for a short time. Ideally, an optimized procedure should be devised, combining in vitro and in vivo systems, in order to achieve synchronization in cattle. We transferred gametes as well as embryos in various stages of development and placed them into the oviducts. Embryos were recovered on Day 7 by flushing of oviducts and uterine horns. Blastocyst rates were determined on Day 7 and on Day 8. Experimental designs included transfer of in vitro matured cumulus oocyte complexes into previously inseminated heifers (COCs group), transfer of in vitro matured COCs simultaneously with capacitated spermatozoa (GIFTs group), transfer of four to eight cell stage embryos developed in vitro after IVM/IVF (Cleaved Stages group) and a group of solely in vitro produced embryos (IVP control group). Our results indicate that in vivo culture of IVM/IVF embryos in the homologous bovine oviduct has a positive influence on subsequent pre-implantation development. In addition, we have evidence that in vitro maturation and in vivo fertilization cannot be synchronized.

Our knowledge of the genes active during normal preimplantation development in cattle is limited, despite the importance for further improvement of fertility and applicability of biotechniques, like in vitro production and embryo transfer. We report on the construction of cDNA libraries as a source for expression profiling in oocytes and single preimplantation cattle embryos. cDNAs were prepared from two unfertilized oocytes, single two-cell, four-cell and eight-cell, morula, and blastocyst stage embryos, respectively. The oocytes, eight-cell, morula, and blastocyst stage embryo-derived cDNAs were ligated to a lambda-based expression vector and these have complexities of 8 x 10(5), 5 x 10(5), 1 x 10(6) and 2 x 10(6) independent clones, respectively. A total of 48 clones were picked and sequenced, 62.5% (30/48) of the sequence were homologous to known transcripts from human and mouse, 18.75% (9/48) to expressed sequence tags (ESTs) of human and mouse origin. Novel sequences were detected at a frequency of 14.58% (7/48). PCR analyses of the embryonic libraries for specific genes revealed transcripts for genes including housekeeping genes (GAPDH and beta-actin), developmental genes (OCT-4, IGF-I receptor and homeodomain sequences) and genes coding for metabolic and protective enzymes (manganese superoxide dismutase, glutamine synthetase, flavin-containing mono-oxygenase, glutamate dehydrogenase, alpha-2-macroglobulin). These cDNA libraries are a valuable resource for the isolation of clones representing genes active at these early developmental stages. The ability to construct cDNA expression libraries from only a few cells will allow gene expression analyses from embryo biopsies and embryos derived by nuclear transfer procedures.