Effects of one-hour daily outdoor access on milk yield and composition and behaviors of tethered dairy cows.

Objective: We investigated the effects of outdoor access for one-hour per day (ODA) on milk yield and composition and behaviors of tethered dairy cows. Methods: Eleven all-day tethered dairy cows were treated the ODA for two weeks. To evaluate the effect of ODA on milk yield, we first calculated the average daily milk yield of each cow for three days during two weeks before the ODA, three days before the ODA, three days at the end of the ODA, and three days during two weeks after the ODA. We then compared the milk yield change during the ODA with that for two weeks before and two weeks after the ODA. The effects of ODA on milk compositions and behaviors were evaluated by comparing the average values for each composition and behavior for the three days before the ODA and the last three days of the ODA. Results: The decrease of milk yield during the two weeks of ODA was significantly higher than that during the two weeks before ODA (p<0.01). The milk fat rate was significantly higher during ODA than before ODA (p<0.05). Lactose rate was significantly lower during ODA than before ODA (p<0.05). The concentrations of milk urea nitrogen, ketone bodies, and free fatty acids in the milk were significantly higher during ODA than before ODA (p<0.05). The mean total duration per day of lying during ODA was significantly lower than that before ODA (p<0.05). The walking steps per one-hour outdoor access were 158.4 ± 54.7. The social behavior during the one-hour outdoor access of the 11 cows was 53 times/h/herd. . Conclusion: Our results suggested that ODA promotes the express of normal behavior in dairy cows, but even one hour of ODA decreases milk production in cows, which may drop producers' profits without some financial supplementation.

Dwell Time Prolongation and Identification of Single Nucleotides Passing through a Solid-State Nanopore by Using Ammonium Sulfate Aqueous Solution.

The ionic current blockades when poly(dT)60 or dNTPs passed through SiN nanopores in an aqueous solution containing (NH4)2SO4 were investigated. The dwell time of poly(dT)60 in the nanopores in an aqueous solution containing (NH4)2SO4 was significantly longer compared to that in an aqueous solution that did not contain (NH4)2SO4. This dwell time prolongation effect due to the aqueous solution containing (NH4)2SO4 was also confirmed when dCTP passed through the nanopores. In addition, when the nanopores were fabricated via dielectric breakdown in the aqueous solution containing (NH4)2SO4, the dwell time prolongation effect for dCTP still occurred even after the aqueous solution was displaced with the aqueous solution without (NH4)2SO4. Furthermore, we measured the ionic current blockades when the four types of dNTPs passed through the same nanopore, and the four types of dNTPs could be statistically identified according to their current blockade values.

Effects of one hour daily outdoor access on lying and sleeping postures, and immune traits of tethered cows.

OBJECTIVE: We investigated the effects of outdoor access for 1 h per day on the animal welfare (AW) of tethered cows, in terms of lying and sleeping postures, and immune function. METHODS: A total of five dry cows were tethered all day indoors (tethering) for 30 days and then tethered indoors with 1 h daily outdoor access (ODA-1h) for 30 days. To analyze the effects of ODA-1h, we calculated the total duration and bout frequency per day, and bout duration of lying and sleeping postures during the last five days of each treatment period. We also analyzed the populations of T cells, B cells, and NK cells in peripheral blood mononuclear cells (PBMC) by fluorescence-activated cell sorting and determined the concanavalin A (Con A) -induced proliferation rate of T cells. RESULTS: The mean total time per day of lying during the ODA-1h treatment was significantly shorter than that during the tethering treatment (p<0.001). The Con A-induced proliferation rate of T cells during the ODA-1h treatment was significantly higher than that during the tethering treatment (p = 0.007). The proportion of NK cells in PBMC during the ODA-1h treatment tended to be higher than that during the tethering treatment (p = 0.062). CONCLUSION: Although ODA-1h may decrease lying time, it increases the available space for tethered cows towards that typically found in grazing and free barn feeding systems. This increased available space promotes the expression of normal behaviors such as walking and social behaviors except lying and may also improve the immune function of tethered dry cows, thereby improving their overall welfare.

Meat the challenge: Segmentation and profiling of Japanese beef mince and its substitutes consumers.

Shifts in protein production methods are an emerging challenge toward realizing a sustainable society. This paper aims to examine preferences among Japanese consumers regarding attributes of beef mince and its substitutes, to develop consumer segments based on these preferences, and to explore the segment with higher acceptance of replacement from conventional products. This paper also aims to explain intersegment differences from consumer heterogeneity in human values, scientific literacy, and sociodemographic viewpoints for a deeper understanding of consumer behavior in each segment. The results of an online choice experiment involving 4421 consumers in Japan, using food labels on mince showed that Japanese-origin organic beef was associated with the highest utility among the five production methods mentioned. Five consumer segments were identified with latent class analysis: novelty accepters, generous customers, attribute-economy balancers, price-conscious, and conservatives, which vary in preference in choice behavior, sociodemographic, human values, and scientific literacy.

Effect of different cow floor mat on the pressure in anterior knees of Holstein dry cows determined using pressure measurement sheet.

To analyze limb pain in tethered dairy cows, we compared three cow floor mat materials (pasture mat® [PM], KKM rubber mat® [RM], and woodchip [WC]) with soil ground considering the pressure applied to the anterior knee of Holstein cows. Three Holstein dry cows (791.3 ± 44.6 kg, 3.8 ± 1.1 years old) moored at the Education and Research Center of Alpine Field Science of the Faculty of Agriculture, Shinshu University, were tested. Pressure applied to the anterior knee of the cow by four different floor mat materials was compared using a commercially available pressure measurement sheet. The sustained pressures applied to the anterior knee of the test cow were 7.2 ± 1. 5, 29.3 ± 5.1, 17.0 ± 1.4, and 27.7 ± 2.5 MPa in the control, RM, PM, and WC, respectively, showing significant lower values (P < 0.05) than in the control. Sustained pressure was significantly lower in the PM than in the other treatments (P < 0.05). The RM was 41.7% cheaper than the PM. The pressure applied to the anterior knee of cow is lower on PM than on RM and WC. PM was more costly than RM but the sustained pressure was much lower on PM.

Effects of starvation-induced negative energy balance on endoplasmic reticulum stress in the liver of cows.

OBJECTIVE: Endoplasmic reticulum (ER) stress engages the unfolded protein response (UPR) that serves as an important mechanism for modulating hepatic fatty acid oxidation and lipogenesis. Chronic fasting in mice induced the UPR activation to regulate lipid metabolism. However, there is no direct evidence of whether negative energy balance (NEB) induces ER stress in the liver of cows. This study aimed to elucidate the relationship between the NEB attributed to feed deprivation and ER stress in bovine hepatocytes. METHODS: Blood samples and liver biopsy tissues were collected from 6 non-lactating cows before and after their starvation for 48 h. The blood non-esterified fatty acids (NEFA), ß-hydroxybutyric acid (BHBA) and glucose level were analyzed. Real-time quantitative polymerase chain reaction and Western blotting were used to explore the regulation of genes associated with UPR and lipid metabolism. RESULTS: The starvation increased the plasma BHBA and NEFA levels and decreased the glucose level. Additionally, the starvation caused significant increases in the mRNA expression level of spliced X-box binding protein 1 (XBP1s) and the protein level of phosphorylated inositol-requiring kinase 1 alpha (p-IRE1α; an upstream protein of XBP1) in the liver. The mRNA expression levels of peroxisome proliferator-activated receptor alpha and its target fatty acid oxidation- and ketogenesis-related genes were significantly upregulated by the starvation-mediated NEB. Furthermore, we found that the mRNA expression levels of lipogenic genes were not significantly changed after starvation. CONCLUSION: These findings suggest that in the initial stage of NEB in dairy cows, the liver coordinates an adaptive response by activating the IRE1 arm of the UPR to enhance ketogenesis, thereby avoiding a fatty liver status.

Sub-10-nm-thick SiN nanopore membranes fabricated using the SiO2sacrificial layer process.

In our previous studies, ultrathin SiN membranes down to 3 nm in thickness were fabricated using the poly-Si sacrificial layer process, and nanopores were formed in those membranes. The region of the SiN membrane fabricated using this process was small, and the poly-Si sacrificial layer remained throughout the other region. On the other hand, to reduce the noise of the current through the nanopore, it is preferable to reduce the capacitance of the nanopore chip by replacing the poly-Si layer with an insulator with low permittivity, such as SiO2. Thus, in this study, the fabrication of SiN membranes with thicknesses of 3-7 nm using the SiO2sacrificial layer process was examined. SiN membranes with thicknesses of less than 5 nm could not be formed when the thickness of the top SiN layer deposited onto the sacrificial layer was 100 nm. In contrast, SiN membranes down to 3.07 nm in thickness could be formed when the top SiN layer was 40 nm in thickness. This is thought to be due to the difference in membrane stress. Nanopores were then fabricated in the membranes via dielectric breakdown. The current noise of the nanopore membranes was approximately 3/5 that of membranes fabricated using the poly-Si sacrificial layer process. Last, ionic current blockades were measured when poly(dT)60passed through the nanopores, and the effective thickness of the nanopores was estimated based on those current-blockade values. The effective thickness was approximately 4.8 nm when the deposited thickness of the SiN membrane was 6.03 nm. On the other hand, the effective thickness and the deposited thickness were almost the same when the deposited thickness was 3.07 nm. This suggests it became difficult to form a shape in which the thickness of the nanopore edge was thinner than the deposited membrane thickness as the deposited thickness decreased.

Effects of adding a commercial capsaicin fertilizer on the feeding behavior of sika deer (Cervus nippon).

We investigated the inhibitory effect of capsaicin fertilizer on feeding in deer. We tested four captive adult female deer. In Experiment 1, in addition to the treatment (intact) containing only a solid feed (HC), we mixed the fertilizer not containing capsaicin (F) or the capsaicin fertilizer (CF) in the solid feed. In addition, the solid feed was put on a wire net that capsaicin fertilizer was placed 5 cm below (SCF). We investigated their feeding behavior response. In Experiment 2, we changed the amount of substance (fertilizer and capsaicin fertilizer) mixed in the HC. We mixed different amounts (0, 50, 100, and 200 g) of the treatments other than the intact with HC and presented them to the deer, and investigated their feeding behavior response. In Experiment 1, intake in the F and CF decreased (p < .05). In Experiment 2, HC intake was significantly lower in the 100 and 200 g CF (p < .05). However, HC intake relatively increased by the last day in the CF 200 g too. The capsaicin fertilizer decreased the feeding behavior of deer by directly touching the mucous membranes of the deer nose and lips. However, the effects were decreased over time.

Low-frequency noise induced by cation exchange fluctuation on the wall of silicon nitride nanopore.

Nanopore-based biosensors have attracted attention as highly sensitive microscopes for detecting single molecules in aqueous solutions. However, the ionic current noise through a nanopore degrades the measurement accuracy. In this study, the magnitude of the low-frequency noise in the ionic current through a silicon nitride nanopore was found to change depending on the metal ion species in the aqueous solution. The order of the low-frequency noise magnitudes of the alkali metal ionic current was consistent with the order of the adsorption affinities of the metal ions for the silanol surface of the nanopore (Li

Solid-state nanopores towards single-molecule DNA sequencing.

Nanopore DNA sequencing offers a new paradigm owing to its extensive potential for long-read, high-throughput detection of nucleotide modification and direct RNA sequencing. Given the remarkable advances in protein nanopore sequencing technology, there is still a strong enthusiasm in exploring alternative nanopore-sequencing techniques, particularly those based on a solid-state nanopore using a semiconductor material. Since solid-state nanopores provide superior material robustness and large-scale integrability with on-chip electronics, they have the potential to surpass the limitations of their biological counterparts. However, there are key technical challenges to be addressed: the creation of an ultrasmall nanopore, fabrication of an ultrathin membrane, control of the ultrafast DNA speed and detection of four nucleotides. Extensive research efforts have been devoted to resolving these issues over the past two decades. In this review, we briefly introduce recent updates regarding solid-state nanopore technologies towards DNA sequencing. It can be envisioned that emerging technologies will offer a brand new future in DNA-sequencing technology.

Stable fabrication of a large nanopore by controlled dielectric breakdown in a high-pH solution for the detection of various-sized molecules.

For nanopore sensing of various-sized molecules with high sensitivity, the size of the nanopore should be adjusted according to the size of each target molecule. For solid-state nanopores, a simple and inexpensive nanopore fabrication method utilizing dielectric breakdown of a membrane is widely used. This method is suitable for fabricating a small nanopore. However, it suffers two serious problems when attempting to fabricate a large nanopore: the generation of multiple nanopores and the non-opening failure of a nanopore. In this study, we found that nanopore fabrication by dielectric breakdown of a SiN membrane under high-pH conditions (pH ≥ 11.3) could overcome these two problems and enabled the formation of a single large nanopore up to 40 nm in diameter within one minute. Moreover, the ionic-current blockades derived from streptavidin-labelled and non-labelled DNA passing through the fabricated nanopore were clearly distinguished. The current blockades caused by streptavidin-labelled DNA could be identified even when its concentration is 1% of the total DNA.

Silicon nitride nanopore created by dielectric breakdown with a divalent cation: deceleration of translocation speed and identification of single nucleotides.

Nanopore DNA sequencing with a solid-state nanopore requires deceleration of the ultrafast translocation speed of single-stranded DNA (ssDNA). We report an unexpected phenomenon: controlled dielectric breakdown (CBD) with a divalent metal cation, especially Ca2+, provides a silicon nitride nanopore with the ability to decelerate ssDNA speed to 100 µs per base even after solution replacement. This speed is two orders of magnitude slower than that for CBD with a conventional monovalent metal cation. Temperature dependence experiments revealed that the enthalpic barrier for a nanopore created via CBD with Ca2+ is 25-30kBT, comparable to that of a biological nanopore. The slowing effect originates from the strong interaction between ssDNA and divalent cations, which were coated on the sidewall of the nanopore during the CBD process. In addition, we found that the nanopore created via CBD with Ca2+ can decelerate the speed of even single-nucleotide monomers, dNMPs, to 0.1-10 ms per base. The four single nucleotides could be statistically identified according to their blockade currents. Our approach is simple and practical because it simultaneously allows nanopore fabrication, ssDNA deceleration and the identification of nucleotide monomers.

Identification of four single-stranded DNA homopolymers with a solid-state nanopore in alkaline CsCl solution.

DNA sequencing via solid-state nanopores is a promising technique with the potential to surpass the performance of conventional sequencers. However, the identification of all four nucleotide homopolymers with a typical SiN nanopore is yet to be clearly demonstrated because a guanine homopolymer rapidly forms a G-quadruplex in a typical KCl aqueous solution. To address this issue, we introduced an alkaline CsCl aqueous solution, which denatures the G-quadruplex into a single-stranded structure by disrupting the hydrogen-bonding network between the guanines and preventing the binding of the K+ ion to G-quartets. Using this alkaline CsCl solution, we provided a proof-of-principle that single-stranded DNA homopolymers of all four nucleotides could be statistically identified according to their blockade currents with the same single nanopore. We also confirmed that a triblock DNA copolymer of three nucleotides exhibited a trimodal Gaussian distribution whose peaks correspond to those of the DNA homopolymers. Our findings contribute to the development of practical DNA sequencing with a solid-state nanopore.

Two-step breakdown of a SiN membrane for nanopore fabrication: Formation of thin portion and penetration.

For the nanopore sensing of various large molecules, such as probe-labelled DNA and antigen-antibody complexes, the nanopore size has to be customized for each target molecule. The recently developed nanopore fabrication method utilizing dielectric breakdown of a membrane is simple and quite inexpensive, but it is somewhat unsuitable for the stable fabrication of a single large nanopore due to the risk of generating multiple nanopores. To overcome this bottleneck, we propose a new technique called "two-step breakdown" (TSB). In the first step of TSB, a local conductive thin portion (not a nanopore) is formed in the membrane by dielectric breakdown. In the second step, the created thin portion is penetrated by voltage pulses whose polarity is opposite to the polarity of the voltage used in the first step. By applying TSB to a 20-nm-thick SiN membrane, a single nanopore with a diameter of 21-26 nm could be fabricated with a high yield of 83%.

Effect of the proportion of roughage fed as rolled and baled hay on repressing wool-biting behavior in housed sheep.

Wool-biting behavior in housed sheep is a serious animal welfare problem and is difficult to control. Since we have found that sheep fed on rolled hay performed less post-feeding wool-biting than those fed on baled hay, here we mixed these two kinds of hay in four different proportions (0%, 33%, 67% and 100% of rolled hay) to test the effect of rolled hay on repressing wool-biting behavior. We also measured the pulling force needed to remove a piece of rolled hay, baled hay, wool and fresh grass. Wool-biting occurred most frequently in the treatment containing 0% rolled hay; however, there was no significant difference between the other three treatments. The pulling force needed to remove a piece of baled hay was significantly weaker than that for the other three objects; no other significant differences in pulling force were found. Our results suggest that the wool-biting behavior of sheep was due to feeding frustration, which arises when they lack the appropriate oral stimulation from performing their normal foraging movement; this then redirects to the wool on their pen-mates. Feeding sheep rolled hay, even in low quantities, can provide them with appropriate oral stimulation and was effective in repressing wool-biting behavior.

Discrimination of three types of homopolymers in single-stranded DNA with solid-state nanopores through external control of the DNA motion.

To achieve DNA sequencing with solid-state nanopores, the speed of the DNA in the nanopore must be controlled to obtain sequence-specific signals. In this study, we fabricated a nanopore-sensing system equipped with a DNA motion controller. DNA strands were immobilized on a Si probe, and approach of this probe to the nanopore vicinity could be controlled using a piezo actuator and stepper motor. The area of the Si probe was larger than the area of the membrane, which meant that the immobilized DNA could enter the nanopore without the need for the probe to scan to determine the location of the nanopore in the membrane. We demonstrated that a single-stranded DNA could be inserted into and removed from a nanopore in our experimental system. The number of different ionic-current levels observed while DNA remained in the nanopore corresponded to the number of different types of homopolymers in the DNA.

Influence of feed type and its effect on repressing wool-biting behavior in housed sheep.

Sheep sometimes develop an abnormal behavior termed as wool-biting when kept in an indoor system; however, little is known about this behavior. As the provided feed type may affect the foraging behavior and repress abnormal behavior in animals, we tested the effect of feed type on repressing wool-biting behavior in this study. We used hay prepared in three forms, that is hay bales, rolls and cubes. The wool-biting frequency associated with hay bales was significantly higher than that associated with rolls (P < 0.05) and cubes (P < 0.05); however, there was no significant difference between rolls and cubes. For hay rolls, wool-biting significantly decreased after feeding (P < 0.05), suggesting that rolls may provide sheep with appropriate oral stimulation; thus, decreasing the post-feeding oral abnormal behavior. An individual difference of wool-biting behavior between sheep was also detected, and an unexpected bed-eating behavior was found in the hay cube treatment. We suggest that sheep performing movements that are similar to their natural foraging behavior while grazing would repress wool-biting behavior, which happened in hay roll and hay cube treatments. Considering sanitation and animal welfare, providing sheep with hay rolls may provide an easier method to control wool-biting behavior in housed sheep.

Integrated solid-state nanopore platform for nanopore fabrication via dielectric breakdown, DNA-speed deceleration and noise reduction.

The practical use of solid-state nanopores for DNA sequencing requires easy fabrication of the nanopores, reduction of the DNA movement speed and reduction of the ionic current noise. Here, we report an integrated nanopore platform with a nanobead structure that decelerates DNA movement and an insulating polyimide layer that reduces noise. To enable rapid nanopore fabrication, we introduced a controlled dielectric breakdown (CDB) process into our system. DNA translocation experiments revealed that single nanopores were created by the CDB process without sacrificing performance in reducing DNA movement speed by up to 10 µs/base or reducing noise up to 600 pArms at 1 MHz. Our platform provides the essential components for proceeding to the next step in the process of DNA sequencing.

Multichannel detection of ionic currents through two nanopores fabricated on integrated Si3N4 membranes.

Integration of solid-state nanopores and multichannel detection of signals from each nanopore are effective measures for realizing high-throughput nanopore sensors. In the present study, we demonstrated fabrication of Si3N4 membrane arrays and the simultaneous measurement of ionic currents through two nanopores formed in two adjacent membranes. Membranes with thicknesses as low as 6.4 nm and small nanopores with diameters of less than 2 nm could be fabricated using the poly-Si sacrificial-layer process and multilevel pulse-voltage injection. Using the fabricated nanopore membranes, we successfully achieved simultaneous detection of clear ionic-current blockades when single-stranded short homopolymers (poly(dA)60) passed through two nanopores. In addition, we investigated the signal crosstalk and leakage current among separated chambers. When two nanopores were isolated on the front surface of the membrane, there was no signal crosstalk or leakage current between the chambers. However, when two nanopores were isolated on the backside of the Si substrate, signal crosstalk and leakage current were observed owing to high-capacitance coupling between the chambers and electrolysis of water on the surface of the Si substrate. The signal crosstalk and leakage current could be suppressed by oxidizing the exposed Si surface in the membrane chip. Finally, the observed ionic-current blockade when poly(dA)60 passed through the nanopore in the oxidized chip was approximately half of that observed in the non-oxidized chip.

Prevention of Dielectric Breakdown of Nanopore Membranes by Charge Neutralization.

To achieve DNA sequencing using a solid-state nanopore, it is necessary to reduce the electric noise current. The noise current can be decreased by reducing the capacitance (C) of the nanopore device. However, we found that an electric-charge difference (ΔQ) between the electrolyte in one chamber and the electrolyte in another chamber occurred. For low capacitance devices, this electric-charge imbalance can lead to unexpectedly high voltage (ΔV = ΔQ/C) which disrupted the membrane when the two electrolytes were independently poured into the chambers. We elucidated the mechanism for the generation of initial defects and established new procedures for preventing the generation of defects by connecting an electric bypass between the chambers.