Heat Stress as a Barrier to Successful Reproduction and Potential Alleviation Strategies in Cattle.

In recent decades, the adverse effects of global warming on all living beings have been unanimously recognized across the world. A high environmental temperature that increases the respiration and rectal temperature of cattle is called heat stress (HS), and it can affect both male and female reproductive functions. For successful reproduction and fertilization, mature and healthy oocytes are crucial; however, HS reduces the developmental competence of oocytes, which compromises reproduction. HS disturbs the hormonal balance that plays a crucial role in successful reproduction, particularly in reducing the luteinizing hormone and progesterone levels, which leads to severe problems such as poor follicle development with a poor-quality oocyte and problems related to maturity, silent estrus, abnormal or weak embryo development, and pregnancy loss, resulting in a declining reproduction rate and losses for the cattle industry. Lactating cattle are particularly susceptible to HS and, hence, their reproduction rate is substantially reduced. Additionally, bulls are also affected by HS; during summer, semen quality and sperm motility decline, leading to compromised reproduction. In summer, the conception rate is reduced by 20-30% worldwide. Although various techniques, such as the provision of water sprinklers, shade, and air conditioning, are used during summer, these methods are insufficient to recover the normal reproduction rate and, therefore, special attention is needed to improve reproductive efficiency and minimize the detrimental effect of HS on cattle during summer. The application of advanced reproductive technologies such as the production of embryos in vitro, cryopreservation during the hot season, embryo transfer, and timed artificial insemination may minimize the detrimental effects of HS on livestock reproduction and recover the losses in the cattle industry.

Label-free electrochemical detection of glucose and glycated hemoglobin (HbA1c).

Measuring glucose together with glycated hemoglobin (HbA1c) could provide short-term and long-term information of blood glycemic condition after a single treatment. However, it has been a challenge to quantify glucose and HbA1c from single sample drop and strip of electrodes with suitable sensitivity, selectivity, and efficiency. In this paper, we demonstrated a label free & single sample drop electrochemical detection method of glucose and HbA1c by modifying carbon electrodes. Glucose oxidase and capture antibodies (C-Ab) against HbA1c were immobilized onto the first working electrode (WE1) and the second working electrode (WE2) of dual working screen-printed carbon electrodes (DWSPCEs), respectively. WE2 was modified with Gold Nano Flower. After that, 3-mercaptopropionic acid was coated as a linker. Finally, C-Ab was bonded by the linker. The relationship between gold surface area and concentration of HgAuCl4 was evaluated to optimize HbA1c incubation time. Linear calibration curves for glucose concentration (0.02-35 mM), HbA1c concentration (0.01 to 1mgml-1), and HbA1c percentage solution (0-14%) were obtained, with correlation coefficients of 0.99. Sensitivities of the biosensor for glucose and HbA1c were 0.5 µAmm-2mM-1 and 0.09 µAmm-2µg-1ml, respectively. The biosensor also showed proper stability (>93%, 45days) and selectivity (>92%). The efficiency of the proposed biosensor was also compared with those of commercial kits using whole blood samples of diabetic and normal cases. Results of this study demonstrate that this biosensor can measure both glucose and HbA1c electrochemically using label-free methods to overview glycemic conditions of blood samples using cheap and commercially viable dual carbon electrode-based biosensors.

Optimization of a Single Substrate-Based Fluorometric Assay for Glucose and Lactate Measurement to Assess Preimplantation Single Embryo Quality and Blood in Obese Mouse and Clinical Human Samples.

A fluorometric assay for single substrate-based glucose and lactate measurements was demonstrated by adopting an already established protocol for glucose and by optimizing pH, enzyme concentration, substrate concentration, and types of buffers for lactate. Linear calibration curves for glucose and lactate concentrations from 1 to 100 µM were obtained with correlation coefficients (R2) of 0.94 and 0.98, respectively. First, with the optimized protocol, single embryo quality was successfully evaluated. Three different initial stages of embryos (n = 58) were cultured for 24 h, and glycolytic activities were calculated by measuring amounts of glucose consumption and lactate production. Results showed that embryos cultured at a later stage had lower glycolytic activities, implying more developmental activities. Second, glucose and lactate concentrations in blood plasma of diet-induced obese (DIO) mice were measured. Levels of both glucose and lactate in DIO mice were higher than those in normal mice by 2.15 and 3.8 mmol/L, respectively (both p < 0.001). Finally, clinical serum samples were analyzed and categorized into three groups based on their measured glucose concentrations: normal (4.73 ± 0.29 mmol/L), prediabetic (6.49 ± 0.13 mmol/L), and diabetic (11.34 ± 1.36 mmol/L) (p < 0.05). Collectively, this developed technique can be used to select a high-quality embryo for transfer as well as to measure glucose and lactate levels in other biological samples.

A Dual Electrode Biosensor for Glucose and Lactate Measurement in Normal and Prolonged Obese Mice Using Single Drop of Whole Blood.

Understanding the levels of glucose (G) and lactate (L) in blood can help us regulate various chronic health conditions such as obesity. In this paper, we introduced an enzyme-based electrochemical biosensor adopting glucose oxidase and lactate oxidase on two working screen-printed carbon electrodes (SPCEs) to sequentially determine glucose and lactate concentrations in a single drop (~30 µL) of whole blood. We developed a diet-induced obesity (DIO) mouse model for 28 weeks and monitored the changes in blood glucose and lactate levels. A linear calibration curve for glucose and lactate concentrations in ranges from 0.5 to 35 mM and 0.5 to 25 mM was obtained with R-values of 0.99 and 0.97, respectively. A drastic increase in blood glucose and a small but significant increase in blood lactate were seen only in prolonged obese cases. The ratio of lactate concentration to glucose concentration (L/G) was calculated as the mouse's gained weight. The results demonstrated that an L/G value of 0.59 could be used as a criterion to differentiate between normal and obesity conditions. With L/G and weight gain, we constructed a diagnostic plot that could categorize normal and obese health conditions into four different zones. The proposed dual electrode biosensor for glucose and lactate in mouse whole blood showed good stability, selectivity, sensitivity, and efficiency. Thus, we believe that this dual electrode biosensor and the diagnostic plot could be used as a sensitive analytical tool for diagnosing glucose and lactate biomarkers in clinics and for monitoring obesity.

Optimized culture systems for the preimplantation ICR mouse embryos with wide range of EDTA concentrations.

In this study, in vitro preimplantation embryo culture media especially for outbred stock mice (Institute of Cancer Research (ICR)) were optimized with different concentrations of ethylenediaminetetraacetic acid (EDTA). A plot with embryo development rates against EDTA concentrations ranging from 0 to 500 µM showed a unique pattern with two characteristic peaks. Two hundred micromolar was adopted as an optimal concentration of EDTA. The optimized media were also evaluated with two culture systems: conventional large volume culture system (1 ml) and micro-droplet culture system. In the conventional large volume culture system, the blastocyst development rates were compared among three different media (F-10, KSOM and KSOM with the optimized 200 µM EDTA). The rates were 0.4%, 16.7% and 57.6%, respectively. The development rates for the micro-droplet (10 µl) culture system were 73.9%. In conclusion, 200 µM EDTA concentration in 10 µl droplets in the KSOM medium was found as the most suitable culture conditions for ICR mouse embryos, as the blastocyst development rate was higher in the micro-droplet culture system than in the traditional conventional large volume culture system.

Sandwich ELISA-Based Electrochemical Biosensor for Leptin in Control and Diet-Induced Obesity Mouse Model.

Leptin is a peptide hormone produced primarily in adipose tissues. Leptin is considered a biomarker associated with obesity and obesity-mediated diseases. Biosensor detection of leptin in the blood may play a critical role as an indicator of dynamic pathological changes. In this paper, we introduce an electrochemical biosensor that adopts o-Phenylenediamine (oPD) on screen-printed gold electrodes (SPGEs) for detecting the leptin from a mouse model of diet-induced obesity (DIO). A linear calibration curve for the leptin concentration was obtained in the ranges from 0.1 to 20 ng/mL with a lower detection limit of 0.033 ng/mL. The leptin concentration was quantified with HRP (horseradish peroxidase)-catalyzed oxidation of oPD by two voltammetry methods: cyclic voltammetry (CV) and square-wave voltammetry (SWV). The proposed sandwich enzyme-linked immunosorbent assay (ELISA)-based electrochemical biosensor for the leptin in mouse blood serum showed high stability, sensitivity, selectivity, and effectivity compared to the commercial Leptin ELISA measurement. Thus, we believe that this leptin biosensor can be a sensitive analytical tool to detect low-levels of biomarkers in clinics and point-of-care testing (POCT).

Flexible and highly ordered nanopillar electrochemical sensor for sensitive insulin evaluation.

In line with growing interest in obesity management, there has been an increase in the amount of research focused on highly sensitive analysis systems for a small number of biomarers. In this paper, we introduce the highly ordered nanopillar electrode, featuring a high aspect ratio surface area that enables enhanced electron transfer. For fabrication of the flexible electrode, gold was evaporated by electronic beam lithography on polyurethane (PU), which has high flexibility. The fabricated nanopillar is 500 nm in diameter and 1500 nm in height. Based on the highly ordered nanostructure electrode, insulin was selected as a biomarker to monitor the insulin resistance associated with obesity. To effectively analyze the insulin, the self-assembled monolayer chemical was used to introduce the enzyme catalysis-based electrochemical immunoassay, leading to the analysis of the insulin concentration range from 0.1 to 1.0 ng/mL in the real sample. The square wave voltammetry principle was used to measure HRP-based electrochemical signal both electrochemically and quantitatively. Based on the nanostructural properties of significant electrochemical behavior, we successfully analyzed insulin in the plasma sample with high sensitivity (LOD of 0.1 ng/mL) and with high reproducibility (<10%). The obtained sensitivity of nanopillar electrode is approximately 10 times (1020%) greater than that of commercial electrode. The results demonstrated that the nanopillar electrode is suitable for precise and sensitive analysis of low-level biomolecules in medical and commercial fields.

Features of Microsystems for Cultivation and Characterization of Stem Cells with the Aim of Regenerative Therapy.

Stem cells have infinite potential for regenerative therapy thanks to their advantageous ability which is differentiable to requisite cell types for recovery and self-renewal. The microsystem has been proved to be more helpful to stem cell studies compared to the traditional methods, relying on its advantageous feature of mimicking in vivo cellular environments as well as other profitable features such as minimum sample consumption for analysis and multiprocedures. A wide variety of microsystems were developed for stem cell studies; however, regenerative therapy-targeted applications of microtechnology should be more emphasized and gain more attractions since the regenerative therapy is one of ultimate goals of biologists and bioengineers. In this review, we introduce stem cell researches harnessing well-known microtechniques (microwell, micropattern, and microfluidic channel) in view point of physical principles and how these systems and principles have been implemented appropriately for characterizing stem cells and finding possible regenerative therapies. Biologists may gain information on the principles of microsystems to apply them to find solutions for their current challenges, and engineers may understand limitations of the conventional microsystems and find new chances for further developing practical microsystems. Through the well combination of engineers and biologists, the regenerative therapy-targeted stem cell researches harnessing microtechnology will find better suitable treatments for human disorders.

"Universal" vitrification of cells by ultra-fast cooling.

Long-term preservation of live cells is critical for a broad range of clinical and research applications. With the increasing diversity of cells that need to be preserved (e.g. oocytes, stem and other primary cells, genetically modified cells), careful optimization of preservation protocols becomes tedious and poses significant limitations for all but the most expert users. To address the challenge of long-term storage of critical, heterogeneous cell types, we propose a universal protocol for cell vitrification that is independent of cell phenotype and uses only low concentrations of cryoprotectant (1.5 M PROH and 0.5 M trehalose). We employed industrial grade microcapillaries made of highly conductive fused silica, which are commonly used for analytical chemistry applications. The minimal mass and thermal inertia of the microcapillaries enabled us to achieve ultrafast cooling rates up to 4,000 K/s. Using the same low, non-toxic concentration of cryoprotectant, we demonstrate high recovery and viability rates after vitrification for human mammary epithelial cells, rat hepatocytes, tumor cells from pleural effusions, and multiple cancer cell lines.

Long-term outcomes of simple crossover stenting from the left main to the left anterior descending coronary artery.

BACKGROUND/AIMS: Although complex bifurcation stenting in patients with non-left main (LM) bifurcation lesions has not yielded better clinical outcomes than simpler procedures, the utility of complex bifurcation stenting to treat LM bifurcation lesions has not yet been adequately explored. METHODS: In the present study, patients who underwent LM-to-left anterior descending (LAD) coronary artery simple crossover stenting to treat significant de novo distal LM or ostial LAD disease, in the absence of angiographically significant ostial left circumflex (LCX) coronary artery disease, were consecutively enrolled. The frequencies of 3-year major adverse cardiovascular events (MACEs; cardiac death, myocardial infarction, and target lesion revascularization), were analyzed. RESULTS: Of 105 eligible consecutive patients, only 12 (11.4%) required additional procedures to treat ostial LCX disease after main vessel stenting. The mean percentage diameter of ostial LCX stenosis increased from 22.5% ± 15.2% to 32.3% ± 16.3% (p < 0.001) after LM-to-LAD simple crossover stenting. The 3-year incidence of MACEs was 9.7% (cardiac death 2.2%; myocardial infarction 2.2%; target lesion revascularization 8.6%), and that of stent thrombosis 1.1%. Of seven cases (7.5%) requiring restenosis, pure ostial LCX-related repeat revascularization was required by only two. CONCLUSIONS: Simple crossover LM-to-LAD stenting without opening of a strut on the LCX ostium was associated with acceptable long-term clinical outcomes.

Real time culture and analysis of embryo metabolism using a microfluidic device with deformation based actuation.

We report a computerized microfluidic real time embryo culture and assay device that can perform automated periodic analyses of embryo metabolism. This automated program uses a modified "gated injection" scheme (sample injection, reagent mixing, enzyme reaction of 15 min incubation, and sample detection) to sequentially measure fluorescence from sample, reference, and background (without any analyte) every hour. Measurements assessed with reference solutions demonstrated the stability of these microfluidic measurements over a 24 h period. Furthermore, this system was able to measure time dependent nutrient consumption by single or multiple (10) live mouse blastocyst-stage embryos with pmol h(-1) sensitivity. Mechanical deformation-based microfluidic actuation created by computerized movement of Braille pins enables automated fluid pumping and valving sequences without unwanted gravity-driven backflow or exposure to electrical fields as would be required in electrokinetic schemes. The convenient, non-invasive, and automated nature of these assays open the way for the development of integrated microfluidic platforms for practical single embryo culture and real time biochemical analysis to assess embryo viability and select embryos with the greatest implantation potential, thus improving success in clinical assisted reproductive technology laboratories.

Controlled loading of cryoprotectants (CPAs) to oocyte with linear and complex CPA profiles on a microfluidic platform.

Oocyte cryopreservation has become an essential tool in the treatment of infertility by preserving oocytes for women undergoing chemotherapy. However, despite recent advances, pregnancy rates from all cryopreserved oocytes remain low. The inevitable use of the cryoprotectants (CPAs) during preservation affects the viability of the preserved oocytes and pregnancy rates either through CPA toxicity or osmotic injury. Current protocols attempt to reduce CPA toxicity by minimizing CPA concentrations, or by minimizing the volume changes via the step-wise addition of CPAs to the cells. Although the step-wise addition decreases osmotic shock to oocytes, it unfortunately increases toxic injuries due to the long exposure times to CPAs. To address limitations of current protocols and to rationally design protocols that minimize the exposure to CPAs, we developed a microfluidic device for the quantitative measurements of oocyte volume during various CPA loading protocols. We spatially secured a single oocyte on the microfluidic device, created precisely controlled continuous CPA profiles (step-wise, linear and complex) for the addition of CPAs to the oocyte and measured the oocyte volumetric response to each profile. With both linear and complex profiles, we were able to load 1.5 M propanediol to oocytes in less than 15 min and with a volumetric change of less than 10%. Thus, we believe this single oocyte analysis technology will eventually help future advances in assisted reproductive technologies and fertility preservation.

Ultra-rapid vitrification of mouse oocytes in low cryoprotectant concentrations.

The ideal cryopreservation protocol would combine the benefits of slow freezing with the benefits of vitrification. This report describes a method for the ultra-rapid vitrification of oocytes using slush nitrogen in quartz capillaries. The approach minimizes the thermal mass of the vitrification vessel by using open microcapillaries made of highly conductive quartz and achieves cooling rates of 250,000 degrees C/min. The process of vitrification can be optimized by maximizing the rate at which the sample is cooled, which allows for the use of lower cryoprotectant concentrations. Mouse oocytes can be successfully vitrified using 1.5 mol/l 1,2-propanediol and 0.5 mol/l trehalose and achieve survival rates of 90.0%(36/40). Fertilization and blastocyst formation rates of vitrified-warmed and fresh oocytes were not significantly different. A total of 120 blastocysts from each of the vitrified-warmed and fresh oocytes were transferred to surrogate mothers and 23 and 27 offspring were born respectively. All offspring in both groups were healthy, grew and bred normally and gave rise to a second generation of pups. Thus, an ultra-rapid vitrification technique has been developed for mouse oocytes that uses low concentrations of cryoprotectants and slush nitrogen in quartz capillaries, which combines the benefits of slow freezing and vitrification.

Characterization and resolution of evaporation-mediated osmolality shifts that constrain microfluidic cell culture in poly(dimethylsiloxane) devices.

Evaporation is a critical problem when handling submicroliter volumes of fluids. This paper characterizes this problem as it applies to microfluidic cell culture in poly(dimethylsiloxane) (PDMS) devices and provides a practical solution. Evaporation-mediated osmolality shifts through PDMS membranes with varying thicknesses (10, 1, 0.2, or 0.1 mm) were measured over 96 h. Even in humidified cell culture incubators, evaporation through PDMS and associated shifts in the osmolality of culture media was significant and prevented mouse embryo and human endothelial cell growth and development. A simple diffusion model, where the measured diffusion coefficient for PDMS matches reported values of approximately 10-9 m2/s, accounts for these evaporation and osmolality shifts. To overcome this problem, a PDMS-parylene-PDMS hybrid membrane was developed that greatly suppresses evaporation and osmolality shifts, yet possesses thinness and the flexibility necessary to interface with deformation-based microfluidic actuation systems, maintains the clarity for optical microscopy, and enables the successful development of single-cell mouse embryos into blastocysts under static conditions and culture of human endothelial cells under dynamic recirculation of submicroliter volumes of media. These insights and methods demonstrated specifically for embryo and endothelial cell studies will be generally useful for understanding and overcoming evaporation-associated effects in microfluidic cell cultures.

Quantitative measurement and control of oxygen levels in microfluidic poly(dimethylsiloxane) bioreactors during cell culture.

Microfluidic bioreactors fabricated from highly gas-permeable poly(dimethylsiloxane) (PDMS) materials have been observed, somewhat unexpectedly, to give rise to heterogeneous long term responses along the length of a perfused mammalian cell culture channel, reminiscent of physiologic tissue zonation that arises at least in part due to oxygen gradients. To develop a more quantitative understanding and enable better control of the physical-chemical mechanisms underlying cell biological events in such PDMS reactors, dissolved oxygen concentrations in the channel system were quantified in real time using fluorescence intensity and lifetime imaging of an oxygen sensitive dye, ruthenium tris(2,2'-dipyridyl) dichloride hexahydrate (RTDP). The data indicate that despite oxygen diffusion through PDMS, uptake of oxygen by cells inside the perfused PDMS microchannels induces an axial oxygen concentration gradient, with lower levels recorded in downstream regions. The oxygen concentration gradient generated by a balance of cellular uptake, convective transport by media flow, and permeation through PDMS in our devices ranged from 0.0003 (mg/l)/mm to 0.7 (mg/l)/mm. The existence of such steep gradients induced by cellular uptake can have important biological consequences. Results are consistent with our mathematical model and give insight into the conditions under which flux of oxygen through PDMS into the microchannels will or will not contribute significantly to oxygen delivery to cells and also provide a design tool to manipulate and control oxygen for cell culture and device engineering. The combination of computerized microfluidics, in situ oxygen sensing, and mathematical models opens new windows for microphysiologic studies utilizing oxygen gradients and low oxygen tensions.

Effects of peak anomalies with the hydrophilic or hydrophobic properties of reservoirs during serial injection on a capillary electrophoresis microchip.

Several anomalies, e.g., in peak shape, migration time, and baseline drift, all due to pressure-driven backflow, were previously reported to occur during serial injection on capillary electrophoresis (CE) chips. Since these anomalies were worse for polydimethylsiloxane (PDMS) microchips than for glass microchips, reproducible data on PDMS microchips were difficult to obtain. In this paper, we found that these problems were affected by the hydrophilic or hydrophobic properties of the reservoirs on the microchip and demonstrated that these anomalies were reduced by converting the hydrophobic properties of the reservoirs on the PDMS microchip into hydrophilic ones. Thus, compared with hydrophobic reservoirs, hydrophilic reservoirs were suitable for the formation of a stable plug. Several chip designs were suggested to reduce these pressure-driven backflows.