Autosomal dominant ApoA4 mutations present as tubulointerstitial kidney disease with medullary amyloidosis.

Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a chr11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionarily conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, including all 25 individuals with kidney failure. Significantly, 11 of 14 genetically unaffected individuals had an eGFR over 60 ml/min/1.73 m2. Fifteen genetically affected individuals presented with higher plasma ApoA4 concentrations. Kidney pathologic specimens from four individuals revealed amyloid deposits limited to the medulla, with the mutated ApoA4 identified by mass-spectrometry as the predominant amyloid constituent in all three available biopsies. Thus, ApoA4 mutations can cause autosomal dominant medullary amyloidosis, with marked amyloid deposition limited to the kidney medulla and presenting with autosomal dominant CKD with a bland urinary sediment. Diagnosis relies on a careful family history, APOA4 sequencing and pathologic studies.

Obese outbred mice only partially benefit from diet normalization or calorie restriction as preconception care interventions to improve metabolic health and oocyte quality.

STUDY QUESTION: Can diet normalization or a calorie-restricted diet for 2 or 4 weeks be used as a preconception care intervention (PCCI) in Western-type diet-induced obese Swiss mice to restore metabolic health and oocyte quality? SUMMARY ANSWER: Metabolic health and oocyte developmental competence was already significantly improved in the calorie-restricted group after 2 weeks, while obese mice that underwent diet normalization showed improved metabolic health after 2 weeks and improved oocyte quality after 4 weeks. WHAT IS KNOWN ALREADY: Maternal obesity is linked with reduced metabolic health and oocyte quality; therefore, infertile obese women are advised to lose weight before conception to increase pregnancy chances. However, as there are no univocal guidelines and the specific impact on oocyte quality is not known, strategically designed studies are needed to provide fundamental insights in the importance of the type and duration of the dietary weight loss strategy for preconception metabolic health and oocyte quality. STUDY DESIGN, SIZE, DURATION: Outbred female Swiss mice were fed a control (CTRL) or high-fat/high-sugar (HF/HS) diet. After 7 weeks, some of the HF mice were put on two different PCCIs, resulting in four treatment groups: (i) only control diet for up to 11 weeks (CTRL_CTRL), (ii) only HF diet for up to 11 weeks (HF_HF), (iii) switch at 7 weeks from an HF to an ad libitum control diet (HF_CTRL) and (iv) switch at 7 weeks from an HF to a 30% calorie-restricted control diet (HF_CR) for 2 or 4 weeks. Metabolic health and oocyte quality were assessed at 2 and 4 weeks after the start of the intervention (n = 8 mice/treatment/time point). PARTICIPANTS/MATERIALS, SETTING, METHODS: Changes in body weight were recorded. To study the impact on metabolic health, serum insulin, glucose, triglycerides, total cholesterol and alanine aminotransferase concentrations were measured, and glucose tolerance and insulin sensitivity were analyzed at PCCI Weeks 2 and 4. The quality of in vivo matured oocytes was evaluated by assessing intracellular lipid droplet content, mitochondrial activity and localization of active mitochondria, mitochondrial ultrastructure, cumulus cell targeted gene expression and oocyte in vitro developmental competence. MAIN RESULTS AND THE ROLE OF CHANCE: Significant negative effects of an HF/HS diet on metabolic health and oocyte quality were confirmed (P < 0.05). HF_CTRL mice already showed restored body weight, serum lipid profile and glucose tolerance, similar to the CTRL_CTRL group after only 2 weeks of PCCI (P < 0.05 compared with HF_HF) while insulin sensitivity was not improved. Oocyte lipid droplet volume was reduced at PCCI Week 2 (P < 0.05 compared with HF_HF), while mitochondrial localization and activity were still aberrant. At PCCI Week 4, oocytes from HF_CTRL mice displayed significantly fewer mitochondrial ultrastructural abnormalities and improved mitochondrial activity (P < 0.05), while lipid content was again elevated. The in vitro developmental capacity of the oocytes was improved but did not reach the levels of the CTRL_CTRL mice. HF_CR mice completely restored cholesterol concentrations and insulin sensitivity already after 2 weeks. Other metabolic health parameters were only restored after 4 weeks of intervention with clear signs of fasting hypoglycemia. Although all mitochondrial parameters in HF_CR oocytes stayed aberrant, oocyte developmental competence in vitro was completely restored already after 2 weeks of intervention. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, we applied a relevant HF/HS Western-type diet to induce obesity in an outbred mouse model. Nevertheless, physiological differences should be considered when translating these results to the human setting. However, the in-depth study and follow-up of the metabolic health changes together with the strategic implementation of specific PCCI intervals (2 and 4 weeks) related to the duration of the mouse folliculogenesis (3 weeks), should aid in the extrapolation of our findings to the human setting. WIDER IMPLICATIONS OF THE FINDINGS: Our study results with a specific focus on oocyte quality provide important fundamental insights to be considered when developing preconception care guidelines for obese metabolically compromised women wishing to become pregnant. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Research Fund (FWO-SB grant 1S25020N and FWO project G038619N). The authors declare there are no conflicts of interest.

Maternal metabolic health and fertility: we should not only care about but also for the oocyte!

Metabolic disorders due to obesity and unhealthy lifestyle directly alter the oocyte's microenvironment and impact oocyte quality. Oxidative stress and mitochondrial dysfunction play key roles in the pathogenesis. Acute effects on the fully grown oocytes are evident, but early follicular stages are also sensitive to metabolic stress leading to a long-term impact on follicular cells and oocytes. Improving the preconception health is therefore of capital importance but research in animal models has demonstrated that oocyte quality is not fully recovered. In the in vitro fertilisation clinic, maternal metabolic disorders are linked with disappointing assisted reproductive technology results. Embryos derived from metabolically compromised oocytes exhibit persistently high intracellular stress levels due to weak cellular homeostatic mechanisms. The assisted reproductive technology procedures themselves form an extra burden for these defective embryos. Minimising cellular stress during culture using mitochondrial-targeted therapy could rescue compromised embryos in a bovine model. However, translating such applications to human in vitro fertilisation clinics is not simple. It is crucial to consider the sensitive epigenetic programming during early development. Research in humans and relevant animal models should result in preconception care interventions and in vitro strategies not only aiming at improving fertility but also safeguarding offspring health.

Capacity of Trolox to improve the development and quality of metabolically compromised bovine oocytes and embryos invitro during different windows of development.

Trials to improve oocyte developmental competence under metabolic stress by using antioxidants may start before or after oocyte maturation. In the present conceptual study, we aimed to identify the most efficient timing of antioxidant application in relation to a metabolic insult using a bovine invitro embryo production model. Pathophysiological concentrations of palmitic acid (PA) were used to induce metabolic stress during oocyte maturation or embryo development. Trolox (TR; antioxidant) treatment prior to, during or after the PA insult was tested to evaluate the protective, neutralising and rescuing capacity of TR respectively. Changes in embryo developmental competence, mitochondrial activity, reactive oxygen species (ROS) concentrations, blastocyst cell allocation and apoptosis and cell stress-related gene expression were monitored. The improvement in developmental capacity was most obvious when oocytes were preloaded with TR before the PA insult. This protective effect could be explained by the observed combination of increased mitochondrial activity with reduced ROS production. This resulted in blastocysts with normal cell counts and apoptosis, as well as increased nuclear factor erythroid 2-related factor 2 (NRF2) expression (a marker for redox regulatory processes) and normalised the expression of the mitochondrial transcription factor A (TFAM), a marker of mitochondrial biogenesis. These results indicate that 'pretreatment' of oocytes with antioxidants produces embryos that seem to be more resilient to a metabolic stress insult.

Effect of overconditioning on the hepatic global gene expression pattern of dairy cows at the end of pregnancy.

Overconditioning is a risk factor for upregulated pre- and postpartum fat mobilization. Therefore, we hypothesized that overconditioning at the end of pregnancy leads to the accumulation of lipids in the liver and modifications of the hepatic gene expression pattern. The aim of this study was to evaluate the effect of normal- versus overconditioning on the hepatic transcriptomic profile of dairy cows at the end of pregnancy. Ten dry multiparous Holstein cows were killed 2 wk before expected calving. Body condition score (BCS) and backfat thickness (BFT) were evaluated, and blood samples for nonesterified fatty acids (NEFA) were taken before cows were killed. After cows were killed, liver biopsy samples were collected for further assessment of total lipids and RNA sequencing. Five cows were classified as normal-conditioned (median BCS = 3, range 2.75-3.5) and 5 as overconditioned (median BCS = 4, range 4-5). Regression models confirmed that normal-conditioned cows had lower BFT (1.29 ± 0.29 cm; least squares means ± standard error) and serum NEFA (0.16 ± 0.04 mmol/L) in comparison to overconditioned cows (3.14 ± 0.43 cm and 0.38 ± 0.07 mmol/L for BFT and NEFA, respectively). Total liver lipid percentage tended to be lower in normal- versus overconditioned cows (4.63 ± 0.40% and 6.06 ± 0.44%, respectively). In comparison to the mean liver lipid percentage of the normal- and overconditioned cows, 1 overconditioned cow had a relatively low (5.21%) and 1 normal-conditioned cow had a relatively high (6.07%) liver lipid percentage. Differentially expressed genes analysis (edgeR quasi-likelihood method) showed that normal-conditioned cows presented 11 upregulated and 12 downregulated genes in comparison to overconditioned cows. Linear discriminant analysis effects size revealed 133 differentially expressed genes between normal- versus overconditioned cows. Notably, the liver of normal-conditioned cows had upregulated genes associated with liver functionality (ALB, SELENOP, IGF1, and IGF2). On the other hand, overconditioned cows had upregulated genes associated with the acute-phase response (C3, HPX, and, LBP). High basal lipolysis in overconditioned cows at the end of pregnancy increased liver lipid content, and this may alter the hepatic gene expression pattern to a pro-inflammatory state.

Ex vivo culture of mouse skin activates an interleukin 1 alpha-dependent inflammatory response.

Ex vivo culture of mouse and human skin causes an inflammatory response characterized by production of multiple cytokines. We used ex vivo culture of mouse tail skin specimens to investigate mechanisms of this skin culture-induced inflammatory response. Multiplex assays revealed production of interleukin 1 alpha (IL-1α), interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), chemokine C-X-C motif ligand 1 (CXCL1), granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) during skin culture, and quantitative PCR revealed transcripts for these proteins were also increased. Ex vivo cultures of skin from myeloid differentiation primary response 88 deficient mice (Myd88-/- ) demonstrated significantly reduced expression of transcripts for the aforementioned cytokines. The same result was observed with skin from interleukin 1 receptor type 1 deficient mice (Il1r1-/- ). These data suggested the IL-1R1/MyD88 axis is required for the skin culture-induced inflammatory response and led us to investigate the role of IL-1α and IL-1ß (the ligands for IL-1R1) in this process. Addition of IL-1α neutralizing antibody to skin cultures significantly reduced expression of Cxcl1, Il6 and Csf3. IL-1ß neutralization did not reduce levels of these transcripts. These studies suggest that IL-1α promotes the skin the culture-induced inflammatory response.

Altered embryotrophic capacities of the bovine oviduct under elevated free fatty acid conditions: an in vitro embryo--oviduct co-culture model.

Maternal metabolic stress conditions are of growing importance in both human and dairy cattle settings as they can have significant repercussions on fertility. Upregulated lipolysis is a common trait associated with metabolic disorders and results in systemically elevated concentrations of non-esterified fatty acids (NEFAs). The effects of high NEFA concentrations on the follicular environment, oocyte and embryo development is well documented. However, knowledge on the effects of NEFAs within the oviduct, representing the initial embryonic growth environment, is currently lacking. Therefore, the experiments outlined here were designed to obtain fundamental insights into both the direct and indirect interactions between NEFAs, bovine oviductal cells and developing zygotes. Hence, zygotes were co-cultured with NEFA-pre-exposed bovine oviductal cells or subjected to simultaneous NEFA exposure during the co-culture period. The outcome parameters assessed were embryo development with cleavage (48h post insemination (pi)), morula (120-126h pi) and blastocyst (192h pi) rates, as well as morula intracellular lipid content and blastocyst quality using Bodipy and differential staining respectively. Our data suggest a direct embryotoxicity of NEFAs as well as impaired embryo development through a reduced oviductal ability to support and protect early embryo development.

Examining the Potential for Agricultural Benefits from Pollinator Habitat at Solar Facilities in the United States.

Of the many roles insects serve for ecosystem function, pollination is possibly the most important service directly linked to human well-being. However, land use changes have contributed to the decline of pollinators and their habitats. In agricultural landscapes that also support renewable energy developments such as utility-scale solar energy [USSE] facilities, opportunities may exist to conserve insect pollinators and locally restore their ecosystem services through the implementation of vegetation management approaches that aim to provide and maintain pollinator habitat at USSE facilities. As a first step toward understanding the potential agricultural benefits of solar-pollinator habitat, we identified areas of overlap between USSE facilities and surrounding pollinator-dependent crop types in the United States (U.S.). Using spatial data on solar energy developments and crop types across the U.S., and assuming a pollinator foraging distance of 1.5 km, we identified over 3,500 km2 of agricultural land near existing and planned USSE facilities that may benefit from increased pollination services through the creation of pollinator habitat at the USSE facilities. The following five pollinator-dependent crop types accounted for over 90% of the agriculture near USSE facilities, and these could benefit most from the creation of pollinator habitat at existing and planned USSE facilities: soybeans, alfalfa, cotton, almonds, and citrus. We discuss how our results may be used to understand potential agro-economic implications of solar-pollinator habitat. Our results show that ecosystem service restoration through the creation of pollinator habitat could improve the sustainability of large-scale renewable energy developments in agricultural landscapes.

A step towards stereotactic navigation during pelvic surgery: 3D nerve topography.

BACKGROUND: Long-term morbidity after multimodal treatment for rectal cancer is suggested to be mainly made up by nerve-injury-related dysfunctions. Stereotactic navigation for rectal surgery was shown to be feasible and will be facilitated by highlighting structures at risk of iatrogenic damage. The aim of this study was to investigate the ability to make a 3D map of the pelvic nerves with magnetic resonance imaging (MRI). METHODS: A systematic review was performed to identify a main positional reference for each pelvic nerve and plexus. The nerves were manually delineated in 20 volunteers who were scanned with a 3-T MRI. The nerve identifiability rate and the likelihood of nerve identification correctness were determined. RESULTS: The analysis included 61 studies on pelvic nerve anatomy. A main positional reference was defined for each nerve. On MRI, the sacral nerves, the lumbosacral plexus, and the obturator nerve could be identified bilaterally in all volunteers. The sympathetic trunk could be identified in 19 of 20 volunteers bilaterally (95%). The superior hypogastric plexus, the hypogastric nerve, and the inferior hypogastric plexus could be identified bilaterally in 14 (70%), 16 (80%), and 14 (70%) of the 20 volunteers, respectively. The pudendal nerve could be identified in 17 (85%) volunteers on the right side and in 13 (65%) volunteers on the left side. The levator ani nerve could be identified in only a few volunteers. Except for the levator ani nerve, the radiologist and the anatomist agreed that the delineated nerve depicted the correct nerve in 100% of the cases. CONCLUSION: Pelvic nerves at risk of injury are usually visible on high-resolution MRI with dedicated scanning protocols. A specific knowledge of their course and its application in stereotactic navigation is suggested to improve quality of life by decreasing the likelihood of nerve injury.

The effect of elevated non-esterified fatty acid concentrations on bovine spermatozoa and on oocyte in vitro fertilisation.

Elevated non-esterified fatty acid (NEFA) concentrations, present in follicular and oviductal fluid, have been postulated as a causative link between metabolic disorders and subfertility. High NEFA conditions can directly disrupt oocyte maturation and developmental capacity after fertilisation. However, their influence on sperm function and the fertilisation process is not known. This study investigated the fertilisation process under high NEFA conditions. To differentiate between effects on both spermatozoa and oocytes or on spermatozoa only, different experiments were conducted. In the first experiment both gametes were simultaneously incubated during IVF under different conditions: (1) NEFA-free, solvent-free control conditions, (2) solvent control, (3) physiological concentrations of oleic (OA), palmitic (PA) and stearic (SA) acids or (4) pathophysiological concentrations of OA, PA and SA. In the second experiment spermatozoa were incubated (4h) under the same treatment conditions prior to routine IVF. Gamete co-incubation resulted in reduced fertilisation and cleavage rates and increased prevalence of polyspermy. In the second experiment embryo developmental capacity and quality were not affected, although sperm motility and plasma membrane integrity were decreased. In conclusion, lipolytic conditions affected the fertilisation process mainly through an effect on the oocyte. Spermatozoa were still able to fertilise even though these conditions reduced sperm function.

Maternal metabolic stress may affect oviduct gatekeeper function.

We hypothesized that elevated non-esterified fatty acids (NEFA) modify in vitro bovine oviduct epithelial cell (BOEC) metabolism and barrier function. Hereto, BOECs were studied in a polarized system with 24-h treatments at Day 9: (1) control (0 µM NEFA + 0% EtOH), (2) solvent control (0 µM NEFA + 0.45% EtOH), (3) basal NEFA (720 µM NEFA + 0.45% EtOH in the basal compartment) and (4) apical NEFA (720 µM NEFA + 0.45% EtOH in the apical compartment). FITC-albumin was used for monolayer permeability assessment and related to transepithelial electric resistance (TER). Fatty acid (FA), glucose, lactate and pyruvate concentrations were measured in spent medium. Intracellular lipid droplets (LD) and FA uptake were studied using Bodipy 493/503 and immunolabelling of FA transporters (FAT/CD36, FABP3 and CAV1). BOEC-mRNA was retrieved for qRT-PCR. Results revealed that apical NEFA reduced relative TER increase (46.85%) during treatment and increased FITC-albumin flux (27.59%) compared to other treatments. In basal NEFA, FAs were transferred to the apical compartment as free FAs: mostly palmitic and oleic acid increased respectively 56.0 and 33.5% of initial FA concentrations. Apical NEFA allowed no FA transfer, but induced LD accumulation and upregulated FA transporter expression (↑CD36, ↑FABP3 and ↑CAV1). Gene expression in apical NEFA indicated increased anti-apoptotic (↑BCL2) and anti-oxidative (↑SOD1) capacity, upregulated lipid metabolism (↑CPT1, ↑ACSL1 and ↓ACACA) and FA uptake (↑CAV1). All treatments had similar carbohydrate metabolism and oviduct function-specific gene expression (OVGP1, ESR1 and FOXJ1). Overall, elevated NEFAs affected BOEC metabolism and barrier function differently depending on NEFA exposure side. Data substantiate the concept of the oviduct as a gatekeeper that may actively alter early embryonic developmental conditions.

Differential effects of high and low glucose concentrations during lipolysis-like conditions on bovine in vitro oocyte quality, metabolism and subsequent embryo development.

Lipolytic metabolic conditions are traditionally associated with elevated non-esterified fatty acid (NEFA) concentrations, but may also be accompanied by hyperglycaemia in obesity or by hypoglycaemia during a negative energy balance status. Elevated NEFA concentrations disrupt oocyte and embryo development and quality, but little is known about whether the effects of lipolytic conditions on oocyte developmental competence are modulated by glucose availability. To answer this, bovine cumulus-oocyte complexes (COCs) were matured under different conditions: physiological NEFA (72µM) and normal glucose (5.5mM), pathophysiologically high NEFA (420µM) and normal glucose, high NEFA and high glucose (9.9mM), high NEFA and low glucose (2.8mM). Developmental potential, cumulus expansion and metabolism of COCs exposed to high NEFA and low glucose were affected to a greater extent compared with COCs matured under high NEFA and high glucose conditions. High NEFA and high glucose conditions caused a moderate increase in oocyte reactive oxygen species compared with their high NEFA and low glucose or control counterparts. Blastocyst metabolism and the transcriptome of metabolic and oxidative stress-related genes were not affected. However, both lipolytic conditions associated with hyper- or hypoglycaemia led to surviving embryos of reduced quality with regards to apoptosis and blastomere allocation.

The Importance of the Periconception Period: Immediate Effects in Cattle Breeding and in Assisted Reproduction Such as Artificial Insemination and Embryo Transfer.

In livestock breeding, the successful outcome is largely depending on the "periconception environment" which, in a narrow sense, refers to the genital tract, where gametogenesis and embryogenesis occur. During these early stages of development, gametes and embryos are known to be particularly sensitive to alterations in their microenvironment. However, as the microenvironment somehow reflects what is going on in the external world, we must widen our definition of "periconception environment" and refer to all events taking place around the time of conception, including metabolic state and health and nutrition of the dam. In modern dairy cows that have to manage an optimal reproductive performance with continued growth and high milk yield, the periconception period is particularly challenging. The metabolic priority for growth and lactation is known to generate adverse conditions hampering optimal ovarian function, oocyte maturation, and development of embryo/fetus. In addition, by using artificial reproductive technologies (ARTs), gametes and/or embryos of livestock are exposed to unnatural conditions outside the male and female genital tract. Artificial insemination, the most widely used technique, is currently yielding pregnancy rates similar to natural mating, and calves produced by AI are equally viable after natural mating. In contrast, other ART, such as multiple ovulation and embryo transfer, have been reported to induce changes in gene expression and DNA methylation patterns with potential consequences for development.Finally, the "periconceptional" environment has been shown to not only influence the successful establishment of pregnancy but also the long-term health and productivity of the offspring. Hence, the optimization of management around the time of conception might open doors to improve animal production and product quality.

Exposure of bovine oocytes and embryos to elevated non-esterified fatty acid concentrations: integration of epigenetic and transcriptomic signatures in resultant blastocysts.

BACKGROUND: Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, such as DNA methylation, in the offspring. Oocyte maturation and early embryo development coincide with methylation changes and both are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect establishment and maintenance of DNA methylation in oocytes and embryos, subsequently altering transcriptomic profiles and developmental competence of resultant blastocysts. RESULTS: Bovine oocytes and embryos were exposed to different NEFA concentrations in separate experiments. In the first experiment, oocytes were matured in vitro for 24 h in medium containing: 1) physiological ("BASAL") concentrations of oleic (OA), palmitic (PA) and stearic (SA) acid or 2) pathophysiological ("HIGH COMBI") concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. Developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray. DNA methylation data were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to BASAL-exposed counterparts (19.3% compared to 23.2% and 18.2% compared to 25.3%, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to adverse environments. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders. CONCLUSIONS: Overall, high variation in methylation between blastocysts made it difficult to draw conclusions concerning methylation of individual genes, although a clear overview of affected pathways was obtained. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later life observed in offspring from mothers displaying lipolytic disorders.

The effect of a negative energy balance status on ß-carotene availability in serum and follicular fluid of nonlactating dairy cows.

Maternal metabolic pressure due to a cow's negative energy balance (NEB) has a negative effect on oocyte quality as a result of increased oxidative stress. In this study, we hypothesized that a NEB status may negatively affect the availability of ß-carotene (bC, an antioxidant) in the micro-environment of the oocyte or follicular fluid (FF) and that daily bC supplementation can increase bC availability. We aimed to (1) determine the effect of a nutritionally induced NEB on bC concentrations in serum and FF as well as on the presence of bC metabolites, oxidative stress levels, and follicular growth in a nonlactating dairy cow model, and (2) investigate how this effect could be altered by dietary bC supplementation. Six multiparous nonlactating Holstein Friesian cows were subjected to 4 consecutive dietary treatments, 28 d each: (1) 1.2 × maintenance (M) or positive energy balance (PEB) without bC supplement (PEB-bC), (2) 1.2 × M with daily supplement of 2,000mg of bC comparable to the level of bC intake at grazing (PEB+bC), (3) 0.6 × M with 2,000mg of bC (NEB+bC), and (4) 0.6 × M (NEB-bC). At the end of each treatment, estrous cycles were synchronized and blood and FF of the largest follicle were sampled and analyzed for bC, retinol, α-tocopherol, free fatty acids, estradiol, and progesterone. Serum cholesterol, triglycerides, urea, insulin growth factor 1, growth hormone, total antioxidant status (TAS), and red blood cell glutathione (GSH) concentrations were determined as well. All cows lost body weight during both energy restriction periods and showed increased serum free fatty acid concentrations, illustrating a NEB. A dietary induced NEB reduced FF bC, but not plasma bC or plasma and FF retinol concentrations. However, bC and retinol concentrations drastically increased in both fluid compartments after bC supplementation. Follicular diameter was increased in supplemented PEB cows. Energy restriction reduced the TAS and red blood cell GSH, whereas daily bC supplementation could restore GSH concentrations, but not the TAS, to levels present in healthy PEB cows. In conclusion, daily bC supplementation can substantially improve bC and retinol availability in the oocyte's micro-environment, irrespective of the energy balance, which may affect follicular development and oocyte quality in the presence of maternal metabolic stress. This knowledge can be of importance to optimize nutritional strategies in the dairy industry to feed for optimal oocyte quality and fertility.

CT and MR imaging for evaluation of cystic renal lesions and diseases.

Cystic renal lesions are commonly encountered in abdominal imaging. Although most cystic renal lesions are benign simple cysts, complex renal cysts, infectious cystic renal disease, and multifocal cystic renal disease are also common phenomena. The Bosniak classification system provides a useful means of categorizing cystic renal lesions but places less emphasis on their underlying pathophysiology. Cystic renal diseases can be categorized as focal, multifocal, or infectious lesions. Diseases that manifest with focal lesions, such as cystic renal cell carcinoma, mixed epithelial and stromal tumor, and cystic nephroma, are often difficult to differentiate but have differing implications for follow-up after resection. Multifocal cystic renal lesions can be categorized as acquired or heritable. Acquired entities, such as glomerulocystic kidney disease, lithium-induced nephrotoxicity, acquired cystic kidney disease, multicystic dysplastic kidney, and localized cystic renal disease, often have distinct imaging and clinical features that allow definitive diagnosis. Heritable diseases, such as autosomal dominant polycystic kidney disease, von Hippel-Lindau disease, and tuberous sclerosis, are usually easily identified and have various implications for patient management. Infectious diseases have varied imaging appearances, and the possibility of infection must not be overlooked when assessing a cystic renal lesion. A thorough understanding of the spectrum of cystic renal disease will allow the radiologist to make a more specific diagnosis and provide the clinician with optimal recommendations for further diagnostic testing and follow-up imaging.

Interaction between differential gene expression profile and phenotype in bovine blastocysts originating from oocytes exposed to elevated non-esterified fatty acid concentrations.

Maternal metabolic disorders linked to lipolysis are major risk factors for reproductive failure. A notable feature of such disorders is increased non-esterified fatty acid (NEFA) concentrations in the blood, which are reflected in the ovarian follicular fluid. Elevated NEFA concentrations impact on the maturing oocyte and even alter subsequent embryo physiology. The aetiological mechanisms have not been fully elucidated. Therefore, in the present study, bovine in vitro maturing cumulus-oocyte complexes were exposed (24 h) to three different maturation treatments containing: (1) physiological (72 µM) NEFA concentrations (=control); (2) elevated (75 µM) stearic acid (SA) concentrations (=HIGH SA); and (3) elevated (425 µM) NEFA concentrations (=HIGH COMBI). Zygotes were fertilised and cultured following standard procedures. Transcriptomic analyses in resulting Day 7.5 blastocysts revealed that the major pathways affected are related to lipid and carbohydrate metabolism in HIGH COMBI embryos and to lipid metabolism and cell death in HIGH SA embryos. Furthermore, lower glutathione content and a reduced number of lipid droplets per cell were observed in HIGH SA-exposed oocytes and resulting morulae, respectively, compared with their HIGH COMBI-exposed counterparts. Vitrified embryos originating from HIGH SA-exposed oocytes tended to exhibit lower survival rates compared with controls. These data suggest possible mechanisms explaining why females across species suffering lipolytic disorders experience difficulties in conceiving.

Characterization of freshly retrieved preantral follicles using a low-invasive, mechanical isolation method extended to different ruminant species.

Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 µm for primordial follicles, 47 ± 6.3 µm for primary follicles and 67.1 ± 13.1 µm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin-eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

Can Species Distribution Models Aid Bioassessment when Reference Sites are Lacking? Tests Based on Freshwater Fishes.

Recent literature reviews of bioassessment methods raise questions about use of least-impacted reference sites to characterize natural conditions that no longer exist within contemporary landscapes. We explore an alternate approach for bioassessment that uses species site occupancy data from museum archives as input for species distribution models (SDMs) stacked to predict species assemblages of freshwater fishes in Texas. When data for estimating reference conditions are lacking, deviation between richness of contemporary versus modeled species assemblages could provide a means to infer relative biological integrity at appropriate spatial scales. We constructed SDMs for 100 freshwater fish species to compare predicted species assemblages to data on contemporary assemblages acquired by four independent surveys that sampled 269 sites. We then compared site-specific observed/predicted ratios of the number of species at sites to scores from a multimetric index of biotic integrity (IBI). Predicted numbers of species were moderately to strongly correlated with the numbers observed by the four surveys. We found significant, though weak, relationships between observed/predicted ratios and IBI scores. SDM-based assessments identified patterns of local assemblage change that were congruent with IBI inferences; however, modeling artifacts that likely contributed to over-prediction of species presence may restrict the stand-alone use of SDM-derived patterns for bioassessment and therefore warrant examination. Our results suggest that when extensive standardized survey data that include reference sites are lacking, as is commonly the case, SDMs derived from generally much more readily available species site occupancy data could be used to provide a complementary tool for bioassessment.

Penetration of daptomycin into bone and synovial fluid in joint replacement.

Daptomycin exhibits clinical activity in the treatment of infections with Gram-positive organisms, including infections due to methicillin-resistant Staphylococcus aureus. However, little is known about its penetration into bone and synovial fluid. The aim of our study was to assess the penetration of daptomycin into bone and synovial fluid after a single intravenous administration. This study was conducted in 16 patients who underwent knee or hip replacement and received a single intravenous dose of 8 mg of daptomycin per kg of body weight prior to surgery. Plasma daptomycin concentrations were measured 1 h after the end of daptomycin infusion and when bone fragments were removed. Daptomycin concentrations were also measured on bone fragments and synovial fluid collected at the same time during surgery. All samples were analyzed with a diode array-high-performance liquid chromatography (HPLC) method. After a single-dose intravenous infusion, bone daptomycin concentrations were above the MIC of daptomycin for Staphylococcus aureus in all subjects, and the median bone penetration percentage was 9.0% (interquartile range [IQR], 4.4 to 11.4). These results support the use of daptomycin in the treatment of Staphylococcus aureus bone and joint infections.