Follicular metabolic alterations are associated with obesity in mares and can be mitigated by dietary supplementation.

Obesity is a growing concern in human and equine populations, predisposing to metabolic pathologies and reproductive disturbances. Cellular lipid accumulation and mitochondrial dysfunction play an important role in the pathologic consequences of obesity, which may be mitigated by dietary interventions targeting these processes. We hypothesized that obesity in the mare promotes follicular lipid accumulation and altered mitochondrial function of oocytes and granulosa cells, potentially contributing to impaired fertility in this population. We also predicted that these effects could be mitigated by dietary supplementation with a combination of targeted nutrients to improve follicular cell metabolism. Twenty mares were grouped as: Normal Weight [NW, n = 6, body condition score (BCS) 5.7 ± 0.3], Obese (OB, n = 7, BCS 7.7 ± 0.2), and Obese Diet Supplemented (OBD, n = 7, BCS 7.7 ± 0.2), and fed specific feed regimens for ≥ 6 weeks before sampling. Granulosa cells, follicular fluid, and cumulus-oocyte complexes were collected from follicles ≥ 35 mm during estrus and after induction of maturation. Obesity promoted several mitochondrial metabolic disturbances in granulosa cells, reduced L-carnitine availability in the follicle, promoted lipid accumulation in cumulus cells and oocytes, and increased basal oocyte metabolism. Diet supplementation of a complex nutrient mixture mitigated most of the metabolic changes in the follicles of obese mares, resulting in parameters similar to NW mares. In conclusion, obesity disturbs the equine ovarian follicle by promoting lipid accumulation and altering mitochondrial function. These effects may be partially mitigated with targeted nutritional intervention, thereby potentially improving fertility outcomes in the obese female.

Design of a Hand-Held and Battery-Operated Digital Microfluidic Device Using EWOD for Lab-on-a-Chip Applications.

Microfluidics offer many advantages to Point of Care (POC) devices through lower reagent use and smaller size. Additionally, POC devices offer the unique potential to conduct tests outside of the laboratory. In particular, Electro-wetting on Dielectric (EWOD) microfluidics has been shown to be an effective way to move and mix liquids enabling many PoC devices. However, much of the research surrounding these microfluidic systems are focused on a single aspect of the system capability, such as droplet control or a specific new application at the device level using the EWOD technology. Often in these experiments the supporting systems required for operation are bench top equipment such as function generators, power supplies, and personal computers. Although various aspects of how an EWOD device is capable of moving and mixing droplets have been demonstrated at various levels, a complete self-contained and portable lab-on-a-chip system based on the EWOD technology has not been well demonstrated. For instance, EWOD systems tend to use high voltage alternating current (AC) signals to actuate electrodes, but little consideration is given to circuitry size or power consumption of such components to make the entire system portable. This paper demonstrates the feasibility of integrating all supporting hardware and software to correctly operate an EWOD device in a completely self-contained and battery-powered handheld unit. We present results that demonstrate a complete sample preparation flow for deoxyribonucleic acid (DNA) extraction and isolation. The device was designed to be a field deployable, hand-held platform capable of performing many other sample preparation tasks automatically. Liquids are transported using EWOD and controlled via a programmable microprocessor. The programmable nature of the device allows it to be configured for a variety of tests for different applications. Many considerations were given towards power consumption, size, and system complexity which make it ideal for use in a mobile environment. The results presented in this paper show a promising step forward to the portable capability of microfluidic devices based on the EWOD technology.

Equine maternal aging affects oocyte lipid content, metabolic function and developmental potential.

Advanced maternal age is associated with a decline in fertility and oocyte quality. We used novel metabolic microsensors to assess effects of mare age on single oocyte and embryo metabolic function, which has not yet been similarly investigated in mammalian species. We hypothesized that equine maternal aging affects the metabolic function of oocytes and in vitro-produced early embryos, oocyte mitochondrial DNA (mtDNA) copy number, and relative abundance of metabolites involved in energy metabolism in oocytes and cumulus cells. Samples were collected from preovulatory follicles from young (≤14 years) and old (≥20 years) mares. Relative abundance of metabolites in metaphase II oocytes (MII) and their respective cumulus cells, detected by liquid and gas chromatography coupled to mass spectrometry, revealed that free fatty acids were less abundant in oocytes and more abundant in cumulus cells from old vs young mares. Quantification of aerobic and anaerobic metabolism, respectively measured as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in a microchamber containing oxygen and pH microsensors, demonstrated reduced metabolic function and capacity in oocytes and day-2 embryos originating from oocytes of old when compared to young mares. In mature oocytes, mtDNA was quantified by real-time PCR and was not different between the age groups and not indicative of mitochondrial function. Significantly more sperm-injected oocytes from young than old mares resulted in blastocysts. Our results demonstrate a decline in oocyte and embryo metabolic activity that potentially contributes to the impaired developmental competence and fertility in aged females.

Study of Real-Time Spatial and Temporal Behavior of Bacterial Biofilms Using 2-D Impedance Spectroscopy.

OBJECTIVE: The purpose of this paper is to demonstrate the use of 2-D impedance spectroscopy to identify areas of biofilm growth on a CMOS biosensor microelectrode-array. METHODS: This paper presents the design and use of a novel multichannel impedance spectroscopy instrument to allow 2-D spatial and temporal evaluation of biofilm growth. The custom-designed circuits can provide a wide range of frequencies (1 Hz-100 kHz) to allow customization of impedance measurements, as the frequency of interest varies based on the type and state of biofilm under measurement. The device is capable of taking measurements as fast as once per second on the entire set of impedance sensors, allowing real-time observation. It also supports adjustable stimulus voltages. The distance between neighboring sensors is 220 micrometers which provides reasonable spatial resolution for biofilm study. RESULTS: Biofilm was grown on the surface of the chip, occupancy was measured using the new tool, and the results were validated optically using fluorescent staining. The results show that the developed tool can be used to determine the bacterial biofilm presence at a given location. CONCLUSION: This paper confirms that 2-D impedance spectroscopy can be used to measure biofilm occupancy. The new tool developed to perform the measurements was able to display real-time results, and determine biofilm coverage of the array electrodes. SIGNIFICANCE: The system presented in this report is the first fully integrated 2-D EIS measurement system with full software support for capturing biofilm growth dynamics in real-time. Due to its ability to nondestructively monitor biofilms over time, 2-D impedance spectroscopy using a microelectrode-array is a useful tool for studying biofilms.

Design of a multi-sensor platform for integrating extracellular acidification rate with multi-metabolite flux measurement for small biological samples.

Rates of cellular oxygen consumption (OCR) and extracellular acidification (ECAR) are widely used proxies for mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic rate in cell metabolism studies. However, ECAR can result from both oxidative metabolism (carbonic acid formation) and glycolysis (lactate release), potentially leading to erroneous conclusions about metabolic substrate utilization. Co-measurement of extracellular glucose and lactate flux along with OCR and ECAR can improve the accuracy and provide better insight into cellular metabolic processes but is currently not feasible with any commercially available instrumentation. Herein, we present a miniaturized multi-sensor platform capable of real-time monitoring of OCR and ECAR along with extracellular lactate and glucose flux for small biological samples such as single equine embryos. This multiplexed approach enables validation of ECAR resulting from OXPHOS versus glycolysis, and expression of metabolic flux ratios that provide further insight into cellular substrate utilization. We demonstrate expected shifts in embryo metabolism during development and in response to OXPHOS inhibition as a model system for monitoring metabolic plasticity in very small biological samples. Furthermore, we also present a preliminary interference analysis of the multi-sensor platform to allow better understanding of sensor interference in the proposed multi-sensor platform. The capability of the platform is illustrated with measurements of multi-metabolites of single-cell equine embryos for assisted reproduction technologies. However, this platform has a wide potential utility for analyzing small biological samples such as single cells and tumor biopsies for immunology and cancer research applications.