Sperm DNA methylation defects in a new mouse model of the 5,10-methylenetetrahydrofolate reductase 677C>T variant and correction with moderate dose folic acid supplementation.

5,10-Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that plays a key role in providing methyl groups for DNA methylation, including during spermatogenesis. A common genetic variant in humans (MTHFR 677C>T) results in reduced enzyme activity and has been linked to various disorders, including male infertility. A new animal model has been created by reproducing the human equivalent of the polymorphism in mice using CRISPR/Cas9. Biochemical parameters in the Mthfr 677TT mice recapitulate alterations found in MTHFR 677TT men. Our aims were to characterize the sperm DNA methylome of the Mthfr 677CC and TT mice on a control diet (2 mg folic acid/kg diet) and assess the effects of folic acid supplementation (10 mg/kg diet) on the sperm DNA methylome. Body and reproductive organ weights, testicular sperm counts, and histology were examined. DNA methylation in sperm was assessed using bisulfite pyrosequencing and whole-genome bisulfite sequencing (WGBS). Reproductive parameters and locus-specific imprinted gene methylation were unaffected by genotype or diet. Using WGBS, sperm from 677TT mice had 360 differentially methylated tiles as compared to 677CC mice, predominantly hypomethylation (60% of tiles). Folic acid supplementation mostly caused hypermethylation in sperm of males of both genotypes and was found to partially correct the DNA methylation alterations in sperm associated with the TT genotype. The new mouse model will be useful in understanding the role of MTHFR deficiency in male fertility and in designing folate supplementation regimens for the clinic.

Effects of Environmental Enrichments on Welfare and Hepatic Metabolic Regulation of Broiler Chickens.

The aims of this study were to find suitable environmental enrichment (EE) and evaluate the combined effect of two EEs, variable light intensity (VL) lighting program and EH, on mental health and hepatic metabolic regulation in commercial broilers. To find the advantageous EEs for broilers, three different EEs (board, hut, and ramp) were tested in trial 1. EEs were placed and the engagement of birds to EEs, dustbathing behavior, and daily physical activity were observed. Birds treated with huts showed higher engagement than the board- or ramp-treated birds (p < 0.05). The results of dustbathing behavior and daily physical activity indicated that the environmental hut (EH) is the most favorable enrichment for broilers. In the second trial, to test the effect of EHs on mental health and hepatic metabolic conditions, the brain and liver were sampled from the four treatment birds (20 lx_Con, 20 lx_Hut, VL_Con and VL_Hut) on day 42. The lower expression of TPH2 (tryptophan hydroxylase 2) of VL_Hut birds than those of VL_Con and 20 lx_Hut treated birds suggests the combining effect of EHs with the VL lighting program on the central serotonergic homeostasis of broilers. Reduced expressions of TH (tyrosine hydroxylase), GR (glucocorticoid receptor), BDNF (brain-derived neurotrophic factor) of VL_Hut treated birds compared to those of VL_Con and 20 lx_Hut birds suggest lower stress, stress susceptibility, and chronic social stress in VL_Hut treated birds. The expression of CPT1A (carnitine palmitoyl transferase 1) increased over three-fold in the liver of VL_Con birds compared to 20 lx_Con birds (p < 0.05). EHs treatment in VL birds (VL_Hut) significantly decreased CPT1A but not in 20 lx birds (20 lx_Hut). The expression of ACCα (acetyl-CoA carboxylase alpha) was significantly decreased in VL_Con birds compared to 20 lx_Con birds. There was no significant difference in the hepatic FBPase (fructose-1,6-bisphosphatase), GR, and 11ß-HSD1 (11 ß-hydroxysteroid dehydrogenease-1) expression between 20 lx_Con and VL_Con birds, but EHs significantly stimulated GR in 20 lx_Hut birds, and stimulated FBPase and 11ß-HSD1 expression in the VL_Hut birds compared to 20 lx_Con birds, suggesting that the VL lighting program reduced fatty acid synthesis and increased fatty acid ß-oxidation in the broilers' liver and VL_Hut improved the hepatic de novo glucose production. Taken together, the results suggest that the stimulated voluntary activity by EHs in the light-enriched broiler house improved mental health and hepatic metabolic function of broilers and may indicate that the improved hepatic metabolic function contributes to efficient nutritional support for broilers.

Folate Deficiency and/or the Genetic Variant Mthfr677C >T Can Drive Hepatic Fibrosis or Steatosis in Mice, in a Sex-Specific Manner.

SCOPE: Disturbances in one-carbon metabolism contribute to nonalcoholic fatty liver disease (NAFLD) which encompasses steatosis, steatohepatitis, fibrosis, and cirrhosis. The goal is to examine impact of folate deficiency and the Mthfr677C >T variant on NAFLD. METHODS AND RESULTS: This study uses the new Mthfr677C >T mouse model for the human MTHFR677C >T variant. Mthfr677CC and Mthfr677TT mice were fed control diet (CD) or folate-deficient (FD) diets for 4 months. FD and Mthfr677TT alter choline/methyl metabolites in liver and/or plasma (decreased S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio, methyltetrahydrofolate, and betaine; increased homocysteine [Hcy]). FD, with contribution from Mthfr677TT, provokes fibrosis in males. Studies of normal livers reveal alterations in plasma markers and gene expression that suggest an underlying predisposition to fibrosis induced by FD and/or Mthfr677TT in males. These changes are absent or reverse in females, consistent with the sex disparity of fibrosis. Sex-based differences in methylation potential, betaine, sphingomyelin, and trimethylamine-N-oxide (TMAO) levels may prevent fibrogenesis in females. In contrast, Mthfr677TT alters choline metabolism, dysregulates expression of lipid metabolism genes, and promotes steatosis in females. CONCLUSION: This study suggests that folate deficiency predisposes males to fibrosis, which is exacerbated by Mthfr677TT, whereas Mthfr677TT predisposes females to steatosis, and reveal novel contributory mechanisms for these NAFLD-related disorders.

Indoor air levels of polycyclic aromatic compounds (PAC) in public buildings with creosote impregnated constructions - A pilot case study using passive samplers.

Creosote has been used in Sweden as a wood preservative in buildings since the 19th century. These buildings can function as workplaces, homes, and cultural buildings to which the public has access. Creosote contains polycyclic aromatic hydrocarbons (PAH) which are well known carcinogens. To understand exposure and risks in an indoor environment, it is important to determine air levels of parent PAHs as well as the more toxic nitrated and oxygenated PAH derivatives (NPAH, OPAH). This study aims to investigate indoor air levels of polycyclic aromatic compounds (PACs) e.g., PAH, NPAH, OPAH and dibenzothiophenes in buildings containing creosote sources and whether these levels pose a health risk. Four cultural buildings were studied, all located within a radius of 130 m. Two were known to have creosote sources, and two had not. Polyurethane foam passive air samplers (PUF-PAS) were used to indicate possible point sources. PUF-PAS measurements were performed for one month in each building winter and summer. Simultaneously, PAC outdoor level measurements were performed. Buildings with creosote impregnated constructions had notably higher indoor air levels of PAC (31-1200 ng m-3) compared to the two buildings without creosote sources (14-45 ng m-3). The PAH cancer potency (sum of benzo[a]pyrene equivalents (BaPeq)) was more than one order of magnitude higher in the buildings containing creosote impregnated wood compared to reference buildings. The highest value was 5.1 BaPeq ng m-3 which was significantly higher than the outdoor winter measurement (1.3 BaPeq ng m-3). Fluoranthene and phenanthrene, with significant distribution in gas phase, but also several particulate NPAHs contributed significantly to the total cancer risk. Thus, creosote containing buildings can still contaminate the indoor air with PACs despite being over a hundred years old. The PUF-PAS was shown to be a good tool providing quantitative/semiquantitative measures of PACs exposure in indoor microenvironments.

Anaerobutyricum and Subdoligranulum Are Differentially Enriched in Broilers with Disparate Weight Gains.

The intestinal microbiota is critically important for animal health and productivity. However, the influence of the intestinal microbiota on animal growth efficiency remains elusive. This current study was aimed at identifying the intestinal bacteria that are associated with the growth rate of broilers in a commercial production setting. Ross 708 broilers with extremely high, medium, and extremely low body weight (BW) were separately selected for each sex from a house of approximately 18,000 chickens on day 42. The cecal content of each animal was subjected to 16S rRNA gene sequencing for microbiota profiling. Our results indicate that a number of bacteria were differentially enriched among different groups of broilers, with several showing a significant correlation (p < 0.05) with BW in both sexes or in a sex-specific manner. Subdoligranulum was drastically diminished in high-BW birds with a strong negative correlation with BW in both males and females. While one Anaerobutyricum strain showed a positive correlation with BW in both sexes, another strain of Anaerobutyricum was positively correlated with BW only in females. These sex-dependent and -independent bacteria could be targeted for improving the growth efficiency and may also be explored as potential biomarkers for the growth rate of broiler chickens.

Effects of a variable light intensity lighting program on the welfare and performance of commercial broiler chickens.

Our previous variable-light intensity lighting program studies indicate the light intensity preference behavior of broilers for their daily activity including eating and resting. To evaluate the effects of variable-light intensity lighting program on performance and welfare of broilers, four commercial trials were conducted for looking at behaviors, mortality, leg-health, performance, and brain welfare indicator genes including tryptophan hydroxylase 2 and tyrosine hydroxylase (TH), glucocorticoid receptor (GR), brain-derived neurotropic factor (BDNF), and melanopsin (Opn4) gene expression. One-day-old broilers were housed in four commercial broiler houses. Each quadrant (section) of the house was placed with 4,800 chicks. A total of four lighting programs began on day 7 with 5 lux (lx), 20 lx, natural light (NL, 480 lx), and variable light (2-5/40 lx) using LED lights on a 16L:8D photoperiod. In the variable-light house, the number of dustbathing holes was significantly higher than that in natural-light houses and 5-lx and 20-lx houses. Daily physical activities, footpad condition, fear response to novel objects, body weight, feed conversion ratio, and the number of leg-problem induced culled birds were affected by the variable-light intensity lighting program. Expression of tryptophan hydroxylase 2 in the DRN and VTA of variable-light treated birds was lower than that of 5-lx- and 20-lx-treated birds on day 42 (p < 0.05). Higher expression of VTA-TH in 5-lx-treated birds than that in 20-lx-, NL-, and variable-light-treated birds suggests the high stress-susceptibility of 5-lx treated birds. Lower VTA-GR expression in 20-lx- and variable-light-treated birds indicates lower stress than that in NL- and 5-lx-treated birds (p < 0.05). The VTA-BDNF expression of NL-treated birds was 2.5 fold higher than that of 5-lx-, 20-lx-, and variable-light-treated birds (p < 0.05), and variable-light-treated birds showed the lowest level of BDNF expression (p < 0.05), suggesting the chronic social defeat stress in NL-treated birds. The result of VTA-Opn4 expression on day 42 suggests the possible role of VTA-Opn4 in broiler welfare through central light perception. Taken together, the variable-light intensity lighting program increased volunteer natural behaviors and physical activity, which may improve footpad condition and leg health of birds, consequently. Performance data including the increased daily weight gain and the lowered feed conversion ratio and results of brain welfare indicator gene expression showed the beneficial effect of the variable-light intensity lighting program on the performance and welfare of commercial broilers.

The 677C > T variant in methylenetetrahydrofolate reductase causes morphological and functional cerebrovascular deficits in mice.

Vascular contributions to cognitive impairment and dementia (VCID) particularly Alzheimer's disease and related dementias (ADRDs) are increasing; however, mechanisms driving cerebrovascular decline are poorly understood. Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in the folate and methionine cycles. Variants in MTHFR, notably 677 C > T, are associated with dementias, but no mouse model existed to identify mechanisms by which MTHFR677C > T increases risk. Therefore, MODEL-AD created a novel knock-in (KI) strain carrying the Mthfr677C > T allele on the C57BL/6J background (Mthfr677C > T) to characterize morphology and function perturbed by the variant. Consistent with human clinical data, Mthfr677C > T mice have reduced enzyme activity in the liver and elevated plasma homocysteine levels. MTHFR enzyme activity is also reduced in the Mthfr677C > T brain. Mice showed reduced tissue perfusion in numerous brain regions by PET/CT as well as significantly reduced vascular density, pericyte number and increased GFAP-expressing astrocytes in frontal cortex. Electron microscopy revealed cerebrovascular damage including endothelial and pericyte apoptosis, reduced luminal size, and increased astrocyte and microglial presence in the microenvironment. Collectively, these data support a mechanism by which variations in MTHFR perturb cerebrovascular health laying the foundation to incorporate our new Mthfr677C > T mouse model in studies examining genetic susceptibility for cerebrovascular dysfunction in ADRDs.

Healthcare professionals' perceptions of dignity-preserving care for older home-dwelling women with incurable cancer in Norway.

Municipal end-of-life care for older home-dwelling patients with cancer is a complex matter requiring healthcare professionals (HCPs) to recognize gender differences in a social, historical and organizational context. A qualitative approach was chosen to explore and identify HCPs value-based principles and organizational conditions promoting dignity-preserving care practice for these women. HCPs recognized the importance of sheltering the women's identity, their sense of being home and acknowledged their personal preferences as value-based principles, whereas creating a flexible culture of care, establishing a functional professional collaboration and developing individualized plans of care, were crucial organizational conditions influencing the practice of dignity-preserving care.

The effect of group or individualised pelvic floor exercises with or without ultrasonography guidance for urinary incontinence in elderly women - A pilot study.

INTRODUCTION: Pelvic floor exercises combined with patient education has shown to be a promising intervention for women suffering from urinary incontinence. This pilot study investigated the effect of patient education combined with group or individualised pelvic floor exercises, or individualised pelvic floor exercises with ultrasonography guidance. METHODS: Thirty-three elderly women with urinary incontinency completed a block-randomised, assessor-blinded study combining patient education with 12-weeks of pelvic floor exercises either group-based or individual with or without ultrasonography guidance. Urinary incontinence symptoms were assessed using the Incontinence Impact Questionnaire-7 (IQ-7) and Urogenital Distress Inventory-6 (UDI-6). Furthermore, daily fluid intake and number of bathroom visits were recorded. Pelvic floor muscle strength was assessed using a manual squeeze test (Oxford Scale, 6-point). RESULTS: An increase in pelvic floor strength was observed after 12 weeks for both the individual (P = 0.038) and the individual ultrasonography-guided (P = 0.01) exercise groups. However, only the latter group maintained an increased strength at the 24-week follow-up (P = 0.008). Across all groups, the intervention led to a decrease in bathroom visits (P = 0.002) that was maintained at the 24-week follow-up (P < 0.001). The interventions led to a decrease in UDI-6 both after the 12-week intervention (P = 0.009) and at the 24-week follow-up (P = 0.032). CONCLUSIONS: These findings indicate that pelvic floor exercises together with patient education can reduce urogenital distress and bathroom visits without change in fluid intake. Furthermore, when pelvic floor exercises were conducted individually, pelvic floor strength increased, but pelvic floor strength was maintained over time only for individualised pelvic floor exercises with ultrasonography guidance.

Impact of experimentally induced bacterial chondronecrosis with osteomyelitis (BCO) lameness on health, stress, and leg health parameters in broilers.

Stress and lameness negatively affect the health, production, and welfare of broilers. Bacterial chondronecrosis with osteomyelitis (BCO) is a leading cause of stress and lameness in commercial broilers. External changes in skin temperature related to changes in blood flow can be detected with infrared thermography (IRT), offering a noninvasive tool to assess the health of animals. This study compared physiological and noninvasive measures of stress and lameness in clinically healthy and lame male broiler chickens between 25 and 56 d. Birds were raised in pens within separate environmental chambers containing either litter flooring (sound) or wire flooring, with the latter established to induce BCO lameness (lame). Physiological and noninvasive measures of stress and lameness were collected: body weight, (BW), relative bursa weight, core body temperature, corticosterone (CORT) concentrations in serum and feathers, surface temperatures of the head (eye and beak) and leg (hock, shank, and foot) regions by infrared thermography (IRT), leg blood oxygen saturation (leg O2), and BCO lesion severity scores of tibial head necrosis (THN) and femoral head necrosis (FHN). Lame birds exhibited greater FHN and THN lesion severities, core body temperatures, and serum CORT (P < 0.05), but had lower BW, relative bursa weight, leg O2, and IRT surface temperatures of the beak, hock, shank, and foot compared with sound birds (P < 0.05). The difference in THN lesion severity between sound and lame birds decreased with age. Linear relationships between leg O2 with IRT leg surface temperatures were positive and negative between leg O2 with BCO lesion severity (P < 0.05). There were negative correlations between serum CORT with hock, shank and foot temperatures (P < 0.001), indicating that BCO is stressful. These results indicate that birds lame from BCO are stressed, have reduced oxygen saturation of blood in their legs, and that IRT surface temperatures can be used as noninvasive indicators of stress and lameness in broilers.

High folic acid intake increases methylation-dependent expression of Lsr and dysregulates hepatic cholesterol homeostasis.

Food fortification with folic acid and increased use of vitamin supplements have raised concerns about high folic acid intake. We previously showed that high folic acid intake was associated with hepatic degeneration, decreased levels of methylenetetrahydrofolate reductase (MTHFR), lower methylation potential, and perturbations of lipid metabolism. MTHFR synthesizes the folate derivative for methylation reactions. In this study, we assessed the possibility that high folic acid diets, fed to wild-type and Mthfr+/- mice, could alter DNA methylation and/or deregulate hepatic cholesterol homeostasis. Digital restriction enzyme analysis of methylation in liver revealed DNA hypomethylation of a CpG in the lipolysis-stimulated lipoprotein receptor (Lsr) gene, which is involved in hepatic uptake of cholesterol. Pyrosequencing confirmed this methylation change and identified hypomethylation of several neighboring CpG dinucleotides. Lsr expression was increased and correlated negatively with DNA methylation and plasma cholesterol. A putative binding site for E2F1 was identified. ChIP-qPCR confirmed reduced E2F1 binding when methylation at this site was altered, suggesting that it could be involved in increasing Lsr expression. Expression of genes in cholesterol synthesis, transport or turnover (Abcg5, Abcg8, Abcc2, Cyp46a1, and Hmgcs1) was perturbed by high folic acid intake. We also observed increased hepatic cholesterol and increased expression of genes such as Sirt1, which might be involved in a rescue response to restore cholesterol homeostasis. Our work suggests that high folic acid consumption disturbs cholesterol homeostasis in liver. This finding may have particular relevance for MTHFR-deficient individuals, who represent ~10% of many populations.

Mild Choline Deficiency and MTHFD1 Synthetase Deficiency Interact to Increase Incidence of Developmental Delays and Defects in Mice.

Folate and choline are interconnected metabolically. The MTHFD1 R653Q SNP is a risk factor for birth defects and there are concerns that choline deficiency may interact with this SNP and exacerbate health risks. 80-90% of women do not meet the Adequate Intake (AI) for choline. The objective of this study was to assess the effects of choline deficiency on maternal one-carbon metabolism and reproductive outcomes in the MTHFD1-synthetase deficient mouse (Mthfd1S), a model for MTHFD1 R653Q. Mthfd1S+/+ and Mthfd1S+/- females were fed control (CD) or choline-deficient diets (ChDD; 1/3 the amount of choline) before mating and during pregnancy. Embryos were evaluated for delays and defects at 10.5 days gestation. Choline metabolites were measured in the maternal liver, and total folate measured in maternal plasma and liver. ChDD significantly decreased choline, betaine, phosphocholine, and dimethylglycine in maternal liver (p < 0.05, ANOVA), and altered phosphatidylcholine metabolism. Maternal and embryonic genotype, and diet-genotype interactions had significant effects on defect incidence. Mild choline deficiency and Mthfd1S+/- genotype alter maternal one-carbon metabolism and increase incidence of developmental defects. Further study is required to determine if low choline intakes contribute to developmental defects in humans, particularly in 653QQ women.

Broiler stress responses to light intensity, flooring type, and leg weakness as assessed by heterophil-to-lymphocyte ratios, serum corticosterone, infrared thermography, and latency to lie.

Stress and leg weakness are detrimental to broiler production, health, and welfare. Traditional methods to evaluate stress may be stressful to the bird because they are invasive and require handling and restraint. Two studies examined the effects of light intensity and flooring on the following in broilers: 1) traditional methods for assessing stress using heterophil-to-lymphocyte ratios and serum corticosterone (CORT) concentrations, 2) noninvasive measures of stress from infrared thermography (IRT) eye and beak surface temperatures, and 3) latency-to-lie (LTL) test times of birds tested individually and in groups of 5. Day-of-hatch male broiler chicks were placed into 6 pens (N = 120 chicks/pen). At 1 wk, pens were allocated to 3 light intensity treatments (2, 5, or 10 lux). At 4 wk, half of the birds from each pen were moved to a pen with wire flooring and the same light intensity. At 1, 4, 5, and 8 wk, blood samples were collected and IRT images of the heads of 5 clinically healthy broilers from each pen were captured. In study 2, IRT images of the heads of birds that became lame in the wire flooring pens were taken. There were no treatment effects on the LTL times of birds tested in groups or individually (P > 0.05). On day 56 in study 1, birds on wire flooring had elevated heterophil-to-lymphocyte ratios and CORT concentrations (P ≤ 0.002) and depressed IRT eye and beak temperatures (P < 0.0001). In both studies, there were negative correlations between CORT concentrations and IRT beak surface temperatures (P < 0.05). Lame birds had lower IRT eye and beak surface temperatures than sound birds (P ≤ 0.004), and the IRT beak surface temperatures of lame birds were lower than their eye surface temperatures (P = 0.004) in study 2. These studies indicate that the IRT surface temperatures of the eye, and more distinctly of the beak, can be used as sensitive noninvasive indicators of stress.

Moderate Folic Acid Supplementation in Pregnant Mice Results in Behavioral Alterations in Offspring with Sex-Specific Changes in Methyl Metabolism.

Fifteen to 20% of pregnant women may exceed the recommended intake of folic acid (FA) by more than four-fold. This excess could compromise neurocognitive and motor development in offspring. Here, we explored the impact of an FA-supplemented diet (5× FASD, containing five-fold higher FA than recommended) during pregnancy on brain function in murine offspring, and elucidated mechanistic changes. We placed female C57BL/6 mice for one month on control diets or 5× FASD before mating. Diets were maintained throughout pregnancy and lactation. Behavioural tests were conducted on 3-week-old pups. Pups and mothers were sacrificed at weaning. Brains and livers were collected to examine choline/methyl metabolites and immunoreactive methylenetetrahydrofolate reductase (MTHFR). 5× FASD led to hyperactivity-like behavior and memory impairment in 3-week-old pups of both sexes. Reduced MTHFR protein in the livers of FASD mothers and male pups resulted in choline/methyl metabolite disruptions in offspring liver (decreased betaine) and brain (decreased glycerophosphocholine and sphingomyelin in male pups, and decreased phosphatidylcholine in both sexes). These results indicate that moderate folate supplementation downregulates MTHFR and alters choline/methyl metabolism, contributing to neurobehavioral alterations. Our findings support the negative impact of high FA on brain development, and may lead to improved guidelines on optimal folate levels during pregnancy.

Biochemical analysis of patients with mutations in MTHFD1 and a diagnosis of methylenetetrahydrofolate dehydrogenase 1 deficiency.

MTHFD1 is a trifunctional protein containing 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylenetetrahydrofolate dehydrogenase activities. It is encoded by MTHFD1 and functions in the cytoplasmic folate cycle where it is involved in de novo purine synthesis, synthesis of thymidylate and remethylation of homocysteine to methionine. Since the first reported case of severe combined immunodeficiency resulting from MTHFD1 mutations, seven additional patients ascertained through molecular analysis have been reported with variable phenotypes, including megaloblastic anemia, atypical hemolytic uremic syndrome, hyperhomocysteinemia, microangiopathy, infections and autoimmune diseases. We determined the level of MTHFD1 expression and dehydrogenase specific activity in cell extracts from cultured fibroblasts of three previously reported patients, as well as a patient with megaloblastic anemia and recurrent infections with compound heterozygous MTHFD1 variants that were predicted to be deleterious. MTHFD1 protein expression determined by Western blotting in fibroblast extracts from three of the patients was markedly decreased compared to expression in wild type cells (between 4.8 and 14.3% of mean control values). MTHFD1 expression in the fourth patient was approximately 44% of mean control values. There was no detectable methylenetetrahydrofolate dehydrogenase specific activity in extracts from any of the four patients. This is the first measurement of MTHFD1 function in MTHFD1 deficient patients and confirms the previous molecular diagnoses.

Effects of light intensity and dual light intensity choice on plasma corticosterone, central serotonergic and dopaminergic activities in birds, Gallus gallus.

Light intensity plays an important role in the regulation of growth, behavior, reproduction, and welfare of avian species. Light intensity preference behavior has been suggested to be involved in welfare of birds. This study aims to investigate the effects of different light intensity and dual light intensity choice (DLIC) lighting program on plasma corticosterone (CORT), and tryptophan hydroxylase 2 (TPH2, the rate-limiting enzyme of serotonin biosynthesis) and tyrosine hydroxylase (TH, the rate-limiting enzyme of dopamine biosynthesis) gene expression in the brainstem of male chickens. Day old broilers were housed in two commercial houses, and placed in 24 pens. All the treatment groups were provided with 23 h light (L) /1 h dark (D) and 30 lx (lx) light intensity during the first week and then 18L:6D (10 lx) from day 7 to 14. Blood and brain were sampled at 14 days of age (10 lx) before the onset of light treatments. On day 15, four treatments (2, 10, 20, and 100 lx), and DLIC treatment (2/20 lx) were initiated. Samples were collected on days 15, 16, 17, 30 and 41. TPH2 expression in the dorsal raphe nucleus (DRN) and caudal raphe nucleus (CRN) of brainstem, and TPH2 and TH expression in ventral tegmental areas (VTN) of the midbrain were determined by qPCR. Results showed that bright light and DLIC lighting program temporarily attenuated plasma CORT, suggesting the short-term stress attenuating effect of bright light and DLIC lighting program. Differential TPH2 expression in the DRN and CRN observed in the DLIC birds indicate a significant effect of DLIC lighting program on the serotonergic activity in the avian brainstem. At the 41 days of age, the significant downregulation of TPH2 and TH expression occurred in the VTA of DLIC treated birds compared to the other group of birds. Taken together, temporal and spatial regulation of TPH2 and TH expression by DLIC lighting program indicate that compensatory regulation of serotonergic and dopaminergic activities might be involved in the light intensity preference behavior of birds, suggesting a possible beneficial effect of the DLIC lighting program on broiler welfare.

Thermal Micro-Environment during Poultry Transportation in South Central United States.

This observational study was conducted to characterize the thermal micro- climate that broilers experienced in commercial poultry transporters under various weather conditions and typical management practices in the South Central USA. We continuously monitored temperature and relative humidity in 45 interior locations of 28 fully-loaded commercial trailers over 2 year spans from 2015⁻2016 in South Central USA. In the cold season, double boarding of the exterior area of the transport modules maintained temperatures at least 8 °C warmer than ambient temperatures as low as -16 °C. Overall, temperature at all locations decreased as transporters traveled from farms to processing plants during winter trips with double boards. In the hot season, assistance by evaporative cooling during on-farm loading resulted in interior temperatures within ± 2 °C of ambient conditions (up to 36 °C) during road transport. In the summer months, trailers uniformly gained 2 °C as vehicles travelled for 45 min from farms to plants. Apparent equivalent temperatures of the monitored summer trips averaged 80.5 °C, indicating possible heat stress conditions based on the thermal comfort zones defined by literature index values. For longer trips, cooling assistance on the farms may be insufficient to prevent temperatures from rising further into extremely hot conditions in the transporters, leading to a dangerous thermal environment.

Mild Methylenetetrahydrofolate Reductase Deficiency Alters Inflammatory and Lipid Pathways in Liver.

SCOPE: Dietary and genetic folate disturbances can lead to nonalcoholic fatty liver disease (NAFLD). A common variant in methylenetetrahydrofolate reductase (MTHFR 677C→T) causes mild MTHFR deficiency with lower 5-methyltetrahydrofolate for methylation reactions. The goal is to determine whether mild murine MTHFR deficiency contributes to NAFLD-related effects. METHODS AND RESULTS: Wild-type and Mthfr+/- mice, a model for the human variant, are fed control (CD) or high-fat (HFAT) diets for 8 weeks. On both diets, MTHFR deficiency results in decreased S-adenosylmethionine, increased S-adenosylhomocysteine, and decreased betaine with reduced methylation capacity, and changes in expression of several inflammatory or anti-inflammatory mediators (Saa1, Apoa1, and Pon1). On CD, MTHFR deficiency leads to microvesicular steatosis with expression changes in lipid regulators Xbp1s and Cyp7a1. The combination of MTHFR deficiency and HFAT exacerbates changes in inflammatory mediators and introduces additional effects on inflammation (Saa2) and lipid metabolism (Nr1h4, Srebf1c, Ppara, and Crot). These effects are consistent with increased expression of pro-inflammatory HDL precursors and greater lipid accumulation. MTHFR deficiency may enhance liver injury through alterations in methylation capacity, inflammatory response, and lipid metabolism. CONCLUSION: Individuals with the MTHFR variant may be at increased risk for liver disease and related complications, particularly when consuming high-fat diets.

The utility of infrared thermography for evaluating lameness attributable to bacterial chondronecrosis with osteomyelitis.

Bacterial chondronecrosis with osteomyelitis (BCO) is a leading cause of lameness in broilers. Infrared thermography (IRT) is a noninvasive technique for measuring infrared radiation from an object and can be used to evaluate clinical health. Two replicated studies compared the effect of light intensity on broilers grown on a wire flooring model that experimentally increased their susceptibility to and incidence of BCO lameness. Day-of-hatch male broiler chickens were placed into 6 pens on wood shavings litter, and at 1 wk one of 3 light intensity treatments (2, 5, or 10 lux) was allotted. At 4 wk half of the population from each pen was moved to a pen with wire flooring and the same light intensity. At 1, 4, 5, and 8 wk, an IRT image of the legs of 5 clinically healthy broilers from each pen was taken. The right and left proximal femora and tibiae of sound and lame broilers were scored for femoral head necrosis (FHN) and tibial head necrosis (THN) lesion severity. There were minimal effects of light intensity and flooring. In Study 1, but not Study 2, broilers on wire flooring weighed less on day 38 (P = 0.007) and days 57 to 58 (P = 0.003) compared to those on litter. The proportion of broilers that became lame on wire flooring was 52% in Study 1 and 14% in Study 2. The proportion of sound broilers from litter and wire flooring pens with subclinical signs of BCO in their right or left proximal growth plates was over 45% for FHN and 92% for THN, and lame broilers had more severe (P < 0.0001) FHN and THN compared to sound broilers. IRT surface temperatures of the hock joint, shank, and foot were consistently lower (P < 0.0001) in broilers that became lame when compared to sound. Therefore, IRT surface temperatures of broiler leg regions may be useful for detecting lesions attributed to BCO.

An evaluation of methods for measuring stress in broiler chickens.

There are potential advantages for using noninvasive methods instead of conventional approaches for measuring corticosterone (CORT) as a metric of stress. Two studies compared blood heterophil: lymphocyte (H:L) ratios, serum CORT, cecal content CORT (Study 1), colon content CORT (Study 2), and feather CORT concentrations for broiler chicks receiving drinking water with or without added CORT. On day 28, male broilers (N = 140) were provided tap water (Control group) or water containing 20 mg/L of CORT (CORT group) for up to 72 h. Body weight (BW), blood, feather, cecal, and colon content samples were collected from 10 birds from each treatment group at 0, 6, 12, 24, 30, 48, and 72 h. Data were analyzed with a JMP Pro ANOVA. Mean comparisons were analyzed on significant treatment effects using post hoc t-tests to compare control and CORT measures within each sampling time point. Pearson's pairwise correlations for all data were performed. A treatment by time interaction affected all measures in both studies. In both studies, CORT treatment reduced body weight and increased H:L ratios at later time points. In study 1 cecal content CORT concentrations were elevated with CORT treatment after 24 h, whereas in study 2 colon content CORT concentrations were elevated after 6 h. CORT treatment increased body and primary feather CORT and primary feather CORT increased with time in control groups in both studies. Serum CORT exhibited the lowest variability compared with all other sample sources used for determining CORT. Estimates of CORT by different methods were positively correlated. These results indicate that serum CORT concentrations most reliably reflect the CORT status of broilers, and that feather CORT concentrations demonstrated potential for estimating stress in broilers.