Dietary Super-Doses of Cholecalciferol Fed to Aged Laying Hens Illustrates Limitation of 24,25-Dihydroxycholecalciferol Conversion.

Background: Older humans taking high concentrations of vitamin D3 supplementation for a prolonged time may be at risk of vitamin D toxicity. It is unclear how dietary super-doses (10,000 times greater than the requirement) can affect vitamin D3 status in aged animals. Aged laying hens could be a model to compare vitamin D3 supplementation effects with women in peri- or postmenopausal stages of life. Objectives: We investigated the dietary super-dose impacts of cholecalciferol (vitamin D3) on vitamin D3 status in aged laying hens in production. Methods: Forty-eight 68-wk-old Hy-Line Brown laying hens were individually housed in cages with 8 hens per dietary treatment for 11 wk. Hens were randomly assigned to 1 of 6 treatment groups of dietary vitamin D3 supplementation and consumed ad libitum. Supplementation concentrations were 400, 800, 7400, 14,000, 20,000, and 36,000 IU D3/kg of feed. At the end of the study, all hens were sacrificed, and tissue samples and feces were collected. Plasma and egg yolk vitamin D3 metabolites, calcium and phosphorus composition of eggshells, ileal digesta, and feces were measured. Duodenal, ileal, liver, and kidney gene expression levels were also measured. Results: We observed that increasing dietary vitamin D3 increased plasma vitamin D3 and egg yolk vitamin D3 (P < 0.0001 for both sites). We also observed an increase in plasma 24,25-dihydroxycholecalciferol as dietary vitamin D3 concentrations increased (P < 0.0001). The plasma 25-hydroxycholecalciferol:24,25-dihydroxycholecalciferol ratio exhibited an asymptotic relationship starting at the 14,000 IU/kg D3 treatment. Conclusions: Dietary super-doses of vitamin D3 led to greater plasma and egg yolk vitamin D3 concentrations, which shows that aged laying hens can deposit excess vitamin D3 in egg yolk. We suggest future research should explore how 24-hydroxylation mechanisms are affected by vitamin D3 supplementation. Further understanding of 24-hydroxylation can help ascertain ways to reduce the risk of vitamin D toxicity.

Medication Safety in Dialysis Care.

The outpatient dialysis setting presents unique challenges in the medication process. Dialysis staff conduct all steps in the medication process, including transcribing and verifying orders, preparing and administering medications, and monitoring for therapeutic and adverse effects. When addressing best medication practices, consideration should be given to education and resources provided to staff. This article explores the multiple strategies taken by a national dialysis network to support clinical staff and improve patient safety.

Characterization of vaccine-induced immune responses against coccidiosis in broiler chickens.

Coccidiosis, caused by Eimeria protozoan species, is an economically important enteric disease of poultry. Although commercial live vaccines are widely used for disease control, the vaccine-induced protective immune mechanisms are poorly characterized. The present study used a commercial broiler vaccine containing a mixture of E. acervulina, E. maxima, and E. tenella. One-day-old chicks were vaccinated by spray followed by a challenge at 21 days of age with a mixture of wild type Eimeria species via oral gavage. Oocyst shedding, immune gene expression and cellular responses in the spleen and cecal tonsils were measured at pre- (days 14 and 21) and post-challenge (days 24, 28 and 35) time points. Results showed that the oocyst counts were significantly reduced in the vaccinated chickens at post-challenge compared to unvaccinated control group. While the vaccinated birds had a significantly increased toll-like receptor (TLR) 21 gene expression at pre-challenge, the transcription of interferon (IFN)γ, Interleukin (IL)-12 and CD40 genes in spleen and cecal tonsils of these birds was significantly higher at post-challenge compared to unvaccinated chickens. Cellular immunophenotyping analysis found that vaccination led to increased frequency of macrophages and activated T cells (CD8+CD44+ and CD4+CD44+) in the spleen and cecal tonsils at post-challenge. Furthermore, in vitro stimulation of chicken macrophages (MQ-NCSU cells) with purified individual species of E. acervulina, E. maxima, and E. tenella showed a significantly increased expression of TLR21, TLR2 and IFNγ genes as well as nitric oxide production. Collectively, these findings suggest that TLR21 and TLR2 may be involved in the immune cell recognition of Eimeria parasites and that the vaccine can induce a robust macrophage activation leading to a T helper-1 dominated protective response at both local and systemic lymphoid tissues.

Influence of incubation, diet, and sex on avian uncoupling protein expression and oxidative stress in market age broilers following exposure to acute heat stress.

Genetic selection for rapid growth in broilers has inadvertently resulted in increased susceptibility to heat stress, particularly in male birds. Increased oxidative stress associated with hyperthermia may be reduced by avian uncoupling protein (avUCP), which has been proposed to modulate free radical production. However, the relationship between avUCP expression and current heat stress management strategies is unclear. Embryonic acclimation or thermal manipulation (TM) and dietary fat source are 2 heat stress interventions that may alter avUCP expression and oxidative stress, but the literature is inconclusive. The objective of this trial was to investigate the effect of TM and dietary fat source on avUCP gene expression and oxidative damage in the breast meat of market age broilers before and after acute heat challenge. The influence of bird sex was also evaluated as broilers exhibit a high degree of sexual dimorphism in growth and stress susceptibility. Concentration of thiobarbituric acid reactive substances (TBARS) was measured as a marker of oxidative damage. Embryonic TM occurred from incubation d 7 to 16 for 12 h daily at 39.5°C. Dietary treatments were applied during the finisher period using either poultry fat, soya oil, or olive oil supplemented at 4.5% in the diet. Acute heat stress (AHS) occurred on d 43 at 32°C for 4 h. Bird performance was decreased by TM, but no significant differences were noted between dietary fat source treatments. Neither avUCP nor TBARS concentrations were significantly influenced by TM or dietary fat source. Downregulation of avUCP was observed following AHS, concurrent with an increase in TBARS concentration. Male birds exhibited higher levels of both avUCP expression and TBARS compared to females and a significant interaction was noted for heat stress by sex, with avUCP expression being greatest in males prior to AHS. The increase in avUCP expression and TBARS concentrations in male birds may be associated with an increased susceptibility to stress arising from the increased growth rate noted for male broilers.

Significance of single ß-mannanase supplementation on performance and energy utilization in broiler chickens, laying hens, turkeys, sows, and nursery-finish pigs: a meta-analysis and systematic review.

This review will give a brief description of ß-mannans, abundance in feedstuffs, utility of supplemental feed ß-mannanase, and subsequent animal responses. Soybean products and co-products of processing palm, coconut, and guar seeds are the major sources of ß-mannans in poultry and livestock feed. ß-Mannans are linear polymers of mannose residues linked by ß-1,4 glycosidic bonds and their ingestion elicit undesirable and metabolically costly responses. Web of Science was searched to retrieve published studies for meta-analyses of the impact of supplemental ß-mannanase on performance and digestibility in pigs and poultry. The mean difference (MD) between ß-mannanase and control on average daily gain (g/d) was +0.23 (P = 0.013; 95% CI of 0.05; 0.41), +10.8 g/d (P = 0.0005; 95% CI of 6.6; 15.0 g/d), and +20.68 (P < 0.000; 95% CI of 17.15; 24.20 g/d) for broiler chickens, nursery pigs, and grow-finish pigs, respectively. The MD on ß-mannanase improvement on feed conversion (FCR) was -0.02 (P < 0.0001) with 95% CI (-0.03; -0.02) suggesting a 2-to-3-point FCR improvement in broiler chickens. ß-Mannanase improvement on gain to feed (G:F) was +13.8 g/kg (P = 0.027; 2.1; 25.4 g/kg) and +8.77 g/kg (6.32; 11.23 g/kg) in nursery and grow-finish pigs, respectively. ß-Mannanase improved apparent metabolizable energy by 47 kcal/kg (P = 0.0004) with 95% CI (28.8; 65.7 kcal/kg) in broiler chickens. The improvement of gross energy digestibility in pigs was 1.08% unit with 95% CI (0.90; 1.26) translating to the release of between 30.6 and 42.8 kcal/kg of digestible energy. Although data were limited, ß-mannanase improved egg production in laying hens linked to improved energy metabolism in laying hens linked to improved energy metabolism but had no impact on egg quality. Turkeys may be more adversely affected by ß-mannans because of the high protein/amino acids requirements necessitating higher dietary inclusion of soybean meal. However, growth performance and feed efficiency responses of turkeys fed diets supplemented with ß-mannanase were variable. In summary, ß-mannanase supplementation improved performance linked to energy and nutrient utilization. However, the magnitude of response was variable within and between species indicating further application refinement is warranted to achieve consistent efficacy, and improved understanding of the functional contribution of ß-mannans hydrolysis products.

Implications of Vitamin D Research in Chickens can Advance Human Nutrition and Perspectives for the Future.

The risk of vitamin D insufficiency in humans is a global problem that requires improving ways to increase vitamin D intake. Supplements are a primary means for increasing vitamin D intake, but without a clear consensus on what constitutes vitamin D sufficiency, there is toxicity risk with taking supplements. Chickens have been used in many vitamin-D-related research studies, especially studies involving vitamin D supplementation. Our state-of-the-art review evaluates vitamin D metabolism and how the different hydroxylated forms are synthesized. We provide an overview of how vitamin D is absorbed, transported, excreted, and what tissues in the body store vitamin D metabolites. We also discuss a number of studies involving vitamin D supplementation with broilers and laying hens. Vitamin D deficiency and toxicity are also described and how they can be caused. The vitamin D receptor (VDR) is important for vitamin D metabolism; however, there is much more to understand about VDR in chickens. Potential research aims involving vitamin D and chickens should explore VDR mechanisms that could lead to newer insights into VDR. Utilizing chickens in future research to help elucidate vitamin D mechanisms has great potential to advance human nutrition. Finding ways to increase vitamin D intake will be necessary because the coronavirus disease 2019 (COVID-19) pandemic is leading to increased risk of vitamin D deficiency in many populations. Chickens can provide a dual purpose with addressing pandemic-caused vitamin D deficiency: 1) vitamin D supplementation gives chickens added-value with the possibility of leading to vitamin-D-enriched meat and egg products; and 2) using chickens in research provides data for translational research. We believe expanding vitamin-D-related research in chickens to include more nutritional aims in vitamin D status has great implications for developing better strategies to improve human health.

Efficacy of 1-α-Hydroxycholecalciferol Supplementation in Young Broiler Feed Suggests Reducing Calcium Levels at Grower Phase.

Increasing biopotency of cholecalciferol (D3) from vitamin sources is essential in the poultry industry to meet nutritional demands and counter stressors. D3 exhibits hormonal traits and is responsible for calcium (Ca) absorption. 1-α-Hydroxycholecalciferol (1α) is a synthetic form of D3 that has equal efficacy and is cheaper to synthesize than 1,25-dihydroxycholecalciferol (active form of D3), on broilers. However, 1α bypasses a critical regulatory point, the kidney, and may consequently lead to toxicity levels of Ca via Ca absorption. This study examined 1α supplementation in broiler diets with different Ca inclusion levels to determine if 1α at higher Ca levels caused Ca toxicity at starter and grower phases with Ross 708 male broiler chicks. In Experiment 1 (1-15 days of age), chicks were assigned to one of 10 treatment starter diets with five levels of Ca inclusion (0.80, 0.95, 1.10, 1.25, and 1.40%) with or without 1α supplementation (5 µg 1α/kg in feed) and eight replicate cages per treatment. In Experiment 2, chicks were fed common starter diet until 16 days of age, and then they were assigned to one of eight treatment diets with four levels of Ca inclusion (0.54, 0.76, 0.98, or 1.20%) with or without 1α supplementation (5 µg 1α/kg in feed). At the end of both experiments, blood was collected from broilers to determine blood chemistry, including concentrations of vitamin D metabolites. Intestinal tissues were also collected to examine gene expression. In Experiment 1, broilers not fed 1α exhibited a quadratic effect in ionized blood Ca (iCa) as dietary Ca inclusion levels increased; 1α-fed broilers displayed an increase in iCa as Ca inclusion levels increased (p = 0.0002). For Experiment 2, 1α-fed broilers displayed a decrease in 25-hydroxycholecalciferol plasma concentration as dietary Ca inclusion levels increased (p = 0.035); also, increasing Ca inclusion in diets increased expression of duodenal sodium phosphate cotransporter type II b (NPTIIb, p = 0.03). Our findings imply that inclusion of 1α was beneficial because 1α enhanced Ca absorption during the starter phase; however, to avoid potential Ca toxicity or antagonism while using 1α during the grower phase, there should be consideration with reducing dietary Ca levels.

Evaluation via Supervised Machine Learning of the Broiler Pectoralis Major and Liver Transcriptome in Association With the Muscle Myopathy Wooden Breast.

The muscle myopathy wooden breast (WB) has recently appeared in broiler production and has a negative impact on meat quality. WB is described as hard/firm consistency found within the pectoralis major (PM). In the present study, we use machine learning from our PM and liver transcriptome dataset to capture the complex relationships that are not typically revealed by traditional statistical methods. Gene expression data was evaluated between the PM and liver of birds with WB and those that were normal. Two separate machine learning algorithms were performed to analyze the data set including the sequential minimal optimization (SMO) of support vector machines (SVMs) and Multilayer Perceptron (MLP) Artificial Neural Network (ANN). Machine learning algorithms were compared to identify genes within a gene expression data set of approximately 16,000 genes for both liver and PM, which can be correctly classified from birds with or without WB. The performance of both machine learning algorithms SMO and MLP was determined using percent correct classification during the cross-validations. By evaluating the WB transcriptome datasets by 5× cross-validation using ANNs, the expression of nine genes ranked based on Shannon Entropy (Information Gain) from PM were able to correctly classify if the individual bird was normal or exhibited WB 100% of the time. These top nine genes were all protein coding and potential biomarkers. When PM gene expression data were evaluated between normal birds and those with WB using SVMs they were correctly classified 95% of the time using 450 of the top genes sorted ranked based on Shannon Entropy (Information Gain) as a preprocessing step. When evaluating the 450 attributes that were 95% correctly classified using SVMs through Ingenuity Pathway Analysis (IPA) there was an overlap in top genes identified through MLP. This analysis allowed the identification of critical transcriptional responses for the first time in both liver and muscle during the onset of WB. The information provided has revealed many molecules and pathways making up a complex molecular mechanism involved with the progression of wooden breast and suggests that the etiology of the myopathy is not limited to activity in the muscle alone, but is an altered systemic pathology.

Effect of Limestone Particle Size and Potassium Supplementation on Growth Performance, Blood Physiology, and Breast Muscle Myopathy of Male Broiler Chickens.

The experiment investigated the effects of limestone particle size and dietary potassium (K) on live performance, blood physiology, and muscle myopathies in broilers raised to 35 days of age. A total of 384 Ross male broilers were placed in 24 floor pens and fed four diets during the starter (0-16 days of age) and grower (17-33 days of age) periods containing two limestone particle sizes (fine: 0.2 mm and coarse: 0.9 mm), and amended with either 0% basal K (K-) or 0.2% added dietary K (K+) as potassium carbonate to complete the 2 × 2 factorial arrangement. Live performance was measured from 1-33 days of age. Blood physiology, woody breast (WB), and white striping (WS) scores were measured at 35 days of age. The K+ dietary treatment reduced (P < 0.05) feed intake and BWG when compared to K- during the starter and grower period. The K+ dietary treatment decreased blood Na (mmol/L), blood glucose (mg/dl), ionized blood Ca (mg/dl), TCO2 (mmol/L), blood HCO3 (mmol/L), and base excess in extracellular fluid (mmol/L) when compared to K- birds of similar body weight at 35 days of age (P ≤ 0.05). Fine limestone diets tended to reduce WB scores (3.0 vs. 2.59) when compared to coarse limestone diets at 35 days of age (P = 0.08). This study demonstrated that using 0.2% of K as potassium carbonate did not negatively affect FCR even though FI and BWG were reduced. Furthermore, fine limestone has the potential to reduce WB in breast muscle tissues; however, further research is needed to confirm these outcomes.

A novel non-invasive method for evaluating electroencephalograms on laying hens.

The use of electroencephalograms (EEG) to study the avian brain relative to behavior was conducted as early as the 1960's. EEG readings, combined with visual cues, provide the ability to elucidate and correlate behaviors to neurological and physiological changes in a chicken. The use of EEG recordings in animal models require access to the brain to implant electrodes. Having the ability to observe EEG activity on sensible birds without surgical implantation could broaden the research in this area and give further insight related to the hen's state of awareness. The development, construction, and implementation of a minimally invasive EEG electrode placement method is described. After implementation, test animals were exposed to extreme environmental stressors as part of a concurrent depopulation methods study and EEG placement withstood the condition changes and corresponding animal physical activity. Sixteen white commercial laying hens had three monopolar 32-gauge needle electrodes inserted subcutaneously and secured to their head and body. Electrodes were attached to a pre-amplifier which transferred EEG signals to a laptop based recording system. Once the electrodes were in place, the hens were placed in individual treatment/observation chamber then various environmental stressors were applied. Verification that the observed brainwave activity was neural and not muscular was done using a photic stimulation validation test. Behavior observations were recorded to correlate sensible and insensible brainwave activity. The validation test and behavior observations demonstrated the method was successful in measuring EEG in sensible laying hens. The use of a non-surgical method for recording EEG will broaden research capabilities and enhance the understanding of a hen's response its environment, eliminate the need for invasive surgical procedures, and minimizes the confounding components of anesthesia, brain surgery, and recovery. With further refinements, the method could open new avenues in avian behavioral and physiological research.

CD4 T-helper cell cytokine phenotypes and antibody response following tetanus toxoid booster immunization.

Routine methods for enumerating antigen-specific T-helper cells may not identify low-frequency phenotypes such as Th2 cells. We compared methods of evaluating such responses to identify tetanus toxoid- (TT) specific Th1, Th2, Th17 and IL10(+) cells. Eight healthy subjects were given a TT booster vaccination. Blood was drawn before, 3, 7, 14, and 28days after vaccination and peripheral blood mononuclear cells (PBMC) were cultured for 7days with TT, negative control (diluent), and a positive control (Staphylococcus enterotoxin B [SEB]). Activation markers (CD25 and CD69) were measured after 44h (n=8), cytokines in supernatant after 3 and 7days, and intracellular cytokine staining (ICS) of proliferated cells (identified by dye dilution) after 7days (n=6). Vaccination increased TT-specific expression of CD25 and CD69 on CD3(+)CD4(+) lymphocytes, and TT-specific proliferation at 7, 14 and 28days post vaccination. Vaccination induced TT-specific Th1 (IFN-γ, TNF-α, and IL-2) Th2 (IL-13, IL-5, and IL-4), Th17 (IL-17A) and IL-10(+) cells as measured by ICS. TT-specific Th1 cells were the most abundant (12-15% of all TT-specific CD4(+) T-cells) while IL10(+) (1.8%) Th17 (1.1%) and Th2 cells (0.2-0.6%) were less abundant. TT-specific cytokine concentrations in PBMC supernatants followed the same pattern where a TT-specific IL-9 response was also seen. In conclusion, TT booster vaccination induced a broad T-helper cell response. This method of evaluating cytokine phenotypes may be useful in examining the impact of nutrition and environmental conditions on the plasticity of T-helper cell memory responses.

Progressive accumulation of epigenetic heterogeneity during human ES cell culture.

Human embryonic stem cells (hESCs) can be maintained in culture over a large number of passages while maintaining apparently normal colony morphology. However, recent reports describe variability in epigenetic states in comparisons among different human ES cell lines. These epigenetic differences include changes in CpG methylation, expression of imprinted genes, and the status of X chromosome inactivation (XCI). We report here that the status of XCI in the female hESC line H9 (WA09) is hypervariable. We find that XIST expression can differ between individual culture isolates of H9. In addition, we find that XIST expression status can vary even between different colonies present within the same H9 culture, effectively rendering the culture mosaic. H9 cultures that lack XIST expression, but have cytological evidence of completed XCI, can also exhibit altered response to BMP4, a growth factor known to induce differentiation of hESCs to a trophectodermal lineage. In the same cultures we find biallelic expression of X-linked genes suggesting that these lines consist of mixtures of cells that retain inactivation of one of two X chromosomes following random choice. Prolonged culture of the XIST-negative isolates to high passage numbers did not result in changes in global epiproteomic signatures, demonstrating rather stable levels of post-translational nucleosome modifications within the culture-adapted hESC lines. The results show that epigenetic variants arise within human ES cell cultures after cell line derivation. In addition, the results indicate that apparently normal cultures of hESCs may contain mixtures of cells with differing epigenetic states. Assays of epigenetic integrity are warranted as quality control measures for the culture of hESCs.

Effects of fgf2 and oxygen in the bmp4-driven differentiation of trophoblast from human embryonic stem cells.

Human embryonic stem cells (hESC) differentiate into trophoblast when treated with BMP4. Here we studied the effects of either low (4 % O(2), L) or atmospheric O(2) (20% O(2), A) in the presence and absence of FGF2 on H1 hESC cultured in presence of BMP4. Differentiation progressed from the periphery towards the center of colonies. It occurred most quickly in the absence of FGF2 and under A and was slowest in presence of FGF2 and under L. Chorionic gonadotrophin (CG) production required A while FGF2 suppressed progesterone synthesis under both A and L. FGF2 was then omitted while we examined trophoblast markers SSEA-1 and cytokeratin-7 and -8, whose expression also progressed inwards from the periphery of colonies and occurred more rapidly under A than L. By day 5, most cells outside central islands of Oct4-positive cells were positive for these antigens under both conditions and many also expressed HLA-G, a marker of extra-villous cytotrophoblast. Under A, but not L, CGalpha and CGbeta became prominent in GATA2-positive, peripherally located, multinucleated cells. In conclusion, BMP4 induced conversion of hESC exclusively towards trophoblast; FGF2 slowed differentiation, while O(2) accelerated this process and promoted syncytiotrophoblast formation.

Striking variation in the sex ratio of pups born to mice according to whether maternal diet is high in fat or carbohydrate.

In female mammals, it remains controversial whether maternal diet and particularly the source and availability of energy can influence sex of offspring born. Outbred female mice were fed ad libitum from 30 days to approximately 45 wk of age on defined, complete diets that differed only in their relative content of fat and carbohydrate to determine whether calorie source influenced litter size and sex ratio of pups. Diet 1 (very high in saturated fat, VHF) provided 60% of calories as fat, mainly lard. Diet 2 (low in saturated fat, LF) was low in fat (10% of calories) but high in carbohydrate. Mice delivered four litters of pups, resulting in a total of 1,048 young born over 108 pregnancies. Gestation length and litter size did not differ between VHF and LF groups and did not change as mice aged. Sex ratio of pups (fraction male) born to mothers on VHF diet was unusually high (0.67) and to mothers on LF diet very low (0.39) over litters 2, 3, and 4. This skewing of sex ratio was related to diets fed and not to body mass of mothers. Age of mothers was an important variable, however. Mice that were first bred at 10 wk of age delivered similar numbers of sons and daughters, whereas virgin mice bred later than 20 wk of age produced litters that were skewed toward males or females according to diet. The data show that the source of calories provided in a nutritionally complete diet to mature female mice can influence sex of offspring born.