Highly sensitive low-frequency-detectable acoustic sensor using a piezoresistive cantilever for health monitoring applications.

This study investigates a cantilever-based pressure sensor that can achieve a resolution of approximately 0.2 mPa, over the frequency range of 0.1-250 Hz. A piezoresistive cantilever with ultra-high acoustic compliance is used as the sensing element in the proposed pressure sensor. We achieved a cantilever with a sensitivity of approximately 40 times higher than that of the previous cantilever device by realizing an ultrathin (340 nm thick) structure with large pads and narrow hinges. Based on the measurement results, the proposed pressure sensor can measure acoustic signals with frequencies as low as 0.1 Hz. The proposed pressure sensor can be used to measure low-frequency pressure and sound, which is crucial for various applications, including photoacoustic-based gas/chemical sensing and monitoring of physiological parameters and natural disasters. We demonstrate the measurement of heart sounds with a high SNR of 58 dB. We believe the proposed microphone will be used in various applications, such as wearable health monitoring, monitoring of natural disasters, and realization of high-resolution photoacoustic-based gas sensors. We successfully measured the first (S1) and second (S2) cardiac sounds with frequencies of 7-100 Hz and 20-45 Hz, respectively.

Technical note: Calving prediction in dairy cattle based on continuous measurements of ventral tail base skin temperature using supervised machine learning.

In this study, we developed a calving prediction model based on continuous measurements of ventral tail base skin temperature (ST) with supervised machine learning and evaluated the predictive ability of the model in 2 dairy farms with distinct cattle management practices. The ST data were collected at 2- or 10-min intervals from 105 and 33 pregnant cattle (mean ± standard deviation: 2.2 ± 1.8 parities) reared in farms A (freestall barn, in a temperate climate) and B (tiestall barn, in a subarctic climate), respectively. After extracting maximum hourly ST, the change in values was expressed as residual ST (rST = actual hourly ST - mean ST for the same hour on the previous 3 d) and analyzed. In both farms, rST decreased in a biphasic manner before calving. Briefly, an ambient temperature-independent gradual decrease occurred from around 36 to 16 h before calving, and an ambient temperature-dependent sharp decrease occurred from around 6 h before until calving. To make a universal calving prediction model, training data were prepared from pregnant cattle under different ambient temperatures (10 data sets were randomly selected from each of the 3 ambient temperature groups: <15°C, ≥15°C to <25°C, and ≥25°C in farm A). An hourly calving prediction model was then constructed with the training data by support vector machine based on 15 features extracted from sensing data (indicative of pre-calving rST changes) and 1 feature from non-sensor-based data (days to expected calving date). When the prediction model was applied to the data that were not part of the training process, calving within the next 24 h was predicted with sensitivities and precisions of 85.3% and 71.9% in farm A (n = 75), and 81.8% and 67.5% in farm B (n = 33), respectively. No differences were observed in means and variances of intervals from the calving alerts to actual calving between farms (12.7 ± 5.8 and 13.0 ± 5.6 h in farms A and B, respectively). Above all, a calving prediction model based on continuous measurement of ST with supervised machine learning has the potential to achieve effective calving prediction, irrespective of the rearing condition in dairy cattle.

Valve-Actuator-Integrated Reference Electrode for an Ultra-Long-Life Rumen pH Sensor.

We demonstrated a newly developed Ag/AgCl reference electrode- with a valve-actuator for two years or longer rumen pH monitoring. Previous studies on pH sensors reported that the short lifetime of Ag/AgCl reference electrodes is caused by an outflow of internal electrolyte. We introduced a valve-actuator into a liquid junction to reduce the outflow by intermittent measurement. The results indicated that the potential change when switching the liquid junction was less than 0.5 mV and its response time was less than 0.083 s. In the 24-h potential measurement with the valve-actuator-integrated reference electrode (VAIRE), the valve was actuated once every hour, and the standard deviation of the potential was 0.29 mV. The lifetime of the VAIRE was estimated at 2.0 years calculating from an electrolyte outflow, which is significantly longer than that of conventional reference electrodes. A pH sensor using the VAIRE was estimated to operate for 2.0 years with the pH error ≤0.1, which meets the requirement of cows' rumen pH monitoring.

Evaluation of ruminal motility in cattle by a bolus-type wireless sensor.

We evaluated the relationship between ruminal motility measured by a force transducer and acceleration measured by bolus sensor, and we assessed the detection of ruminal motility in cattle by a bolus-type wireless sensor. The bolus sensor can be orally administered to cattle and was placed in the reticulum for continuous measurements. The probe was almost horizontal to the longitudinal axis. The bolus sensor's basic y-axis acceleration movement appeared to have a very distinct vertical pattern, occurring roughly 1-1.5 times/min with a duration of approximately 8 sec, displaying at around 500 mG. A significant positive correlation was observed between the ruminal contraction revealed by the force transducer and the acceleration shown by the bolus sensor (P<0.01). The contraction of the dorsal sac of the rumen and the acceleration signals in the reticulum occurred at practically the same time. The frequency and amplitude of ruminal contraction demonstrated by the bolus sensor and the force transducer in feeding were significantly higher than those at rest (P<0.01). The bolus sensor could also detect ruminal atony in the cattle after the administration of xylazine. A bolus-type wireless sensor may thus be useful for the measurement of ruminal motility in cattle and for detecting rumen dysfunction (e.g., ruminal atony).

Estrous detection by continuous measurements of vaginal temperature and conductivity with supervised machine learning in cattle.

This study aimed to evaluate the effectiveness of estrous detection technique based on continuous measurements of vaginal temperature (VT) and conductivity (VC) with supervised machine learning in cattle. The VT and VC of 17 cows in tie-stalls were measured using our developed wearable vaginal sensor from Day 11 (Day 0 = ovulation day) to Day 11 of the subsequent estrous cycle at 15-min interval. After the maximum VT and VC were extracted hourly, their changes were expressed as residual VT (rVT = actual VT - mean VT for the same time on the previous 3 days) and as VC ratio (VCr = actual VC/mean VC for the same time on Day 11-13), respectively, and were used for analysis. Trans-rectal ultrasonography was performed to monitor ovarian structure changes. The plasma concentrations of reproductive hormones (progesterone: P4, estradiol-17ß: E2, and LH) were measured in the experimental period. Standing estrus was confirmed by testing with herd mates at 3-h interval. The rVT decreased transiently, which coincided with decreasing P4 a few days before estrus, and a sharp increase was associated with LH surge during estrus. The VCr increased as estrus approached, corresponding with decreasing P4 and increasing E2 and LH. After noise reduction, features, possible to follow-up estrus-associated changes in rVT and VCr, were extracted and used for developing estrous detection models; 9 models were developed with 3 feature sets (features extracted from rVT alone, VCr alone, and combination of rVT and VCr) and 3 machine learning algorithms (decision tree: DT, support vector machine: SVM, and artificial neural network: ANN). Cross-validation showed that models using the features from the combination of rVT and VCr showed better performance in terms of sensitivity and precision than those using features from VCr alone, and precision than those of using features from rVT alone. Within the models using the features from the combination of rVT and VCr, sensitivity and precision of the model generated by ANN were numerically, but not statistically, higher than those generated by DT and SVM. Of 17 estruses, 16 were detected, with one false positive, when the best model was used. Furthermore, both mean and variance of the interval from the beginning of the estrous detection alert to ovulation (27.3 ±â€¯6.7 h, mean ±â€¯SD of 16 estruses) were not significantly different to those from the onset of standing estrus to ovulation (30.8 ±â€¯5.8 h, n = 17), indicating that the estrus can be detected real-time by the present technique. Hence, the estrous detection technique based on continuous measurements of VT and VC with supervised machine learning has a potential for efficient and accurate estrous detection in cattle.

Estrous detection by monitoring ventral tail base surface temperature using a wearable wireless sensor in cattle.

In the present study, the ventral tail base surface temperature (ST) was monitored using a wearable wireless sensor for estrus detection in cattle. Relationships among ST, behavioral estrus expression, ovulation, and changes in hormone profiles during the estrous cycle were examined. Holstein Friesian or Japanese Black female cattle were used in summer (August-September), autumn (October-November) and winter (January-February; three animals per season). On Day 11 of the estrous cycle (Day 0=the day of ovulation), the sensor was attached to the surface of the ventral tail base and ST was measured every 2min until Day 11 of the next estrous cycle. Hourly maximum ST values were used for analysis. To exclude circadian rhythm and seasonal effects, ST changes were expressed as residual temperatures (RT=actual ST - mean ST for the same hour on the previous 3days). Obvious circadian rhythms of the ST were observed and daily changes in the ST significantly differed among seasons. There was no significant seasonal difference, however, in the RT. The mean RT increased significantly ∼24 compared with ∼48h before ovulation. The mean maximum RT was 1.27±0.30°C, which was observed 5.6±2.4h after the onset of estrus, 2.4±1.3h before LH peak, and 26.9±1.2h before ovulation. The ST of the ventral tail base could be monitored throughout the estrous cycle and could detect a substantial change around the time of expression of behavioral estrus. Calculation and analysis of the RT could be useful for automatic estrous detection.

Minimized Bolus-Type Wireless Sensor Node with a Built-In Three-Axis Acceleration Meter for Monitoring a Cow's Rumen Conditions.

Monitoring rumen conditions in cows is important because a dysfunctional rumen system may cause death. Sub-acute ruminal acidosis (SARA) is a typical disease in cows, and is characterized by repeated periods of low ruminal pH. SARA is regarded as a trigger for rumen atony, rumenitis, and abomasal displacement, which may cause death. In previous studies, rumen conditions were evaluated by wireless sensor nodes with pH measurement capability. The primary advantage of the pH sensor is its ability to continuously measure ruminal pH. However, these sensor nodes have short lifetimes since they are limited by the finite volume of the internal liquid of the reference electrode. Mimicking rumen atony, we attempt to evaluate the rumen condition using wireless sensor nodes with three-axis accelerometers. The theoretical life span of such sensor nodes depends mainly on the transmission frequency of acceleration data and the size of the battery, and the proposed sensor nodes are 30.0 mm in diameter and 70.0 mm in length and have a life span of over 600 days. Using the sensor nodes, we compare the rumen motility of the force transducer measurement with the three-axis accelerometer data. As a result, we can detect discriminative movement of rumen atony.

Development of a wireless sensor for the measurement of chicken blood flow using the laser Doppler blood flow meter technique.

Here, we report the development of an integrated laser Doppler blood flow micrometer for chickens. This sensor weighs only 18 g and is one of the smallest-sized blood flow meters, with no wired line, these are features necessary for attaching the sensor to the chicken. The structure of the sensor chip consists of two silicon cavities with a photo diode and a laser diode, which was achieved using the microelectromechanical systems technique, resulting in its small size and significantly low power consumption. In addition, we introduced an intermittent measuring arrangement in the measuring system to reduce power consumption and to enable the sensor to work longer. We were successfully able to measure chicken blood flow for five consecutive days, and discovered that chicken blood flow shows daily fluctuations.

Emergence of avian influenza viruses with enhanced transcription activity by a single amino acid substitution in the nucleoprotein during replication in chicken brains.

To explore the genetic basis of the pathogenesis and adaptation of avian influenza viruses (AIVs) to chickens, the A/duck/Yokohama/aq10/2003 (H5N1) (DkYK10) virus was passaged five times in the brains of chickens. The brain-passaged DkYK10-B5 caused quick death of chickens through rapid and efficient replication in tissues, accompanied by severe apoptosis. Genome sequence comparison of two viruses identified a single amino acid substitution at position 109 in NP from isoleucine to threonine (NP (I)109(T)). By analyzing viruses constructed by the reverse-genetic method, we established that the NP (I)109(T) substitution also contributed to increased viral replication and polymerase activity in chicken embryo fibroblasts, but not in duck embryo fibroblasts. Real-time RT-PCR analysis demonstrated that the NP (I)109(T) substitution enhances mRNA synthesis quickly and then cRNA and viral RNA (vRNA) synthesis slowly. Next, to determine the mechanism underlying the appearance of the NP (I)109(T) substitution during passages, four H5N1 highly pathogenic AIVs (HPAIVs) were passaged in the lungs and brains of chicken embryos. Single-nucleotide polymorphism analysis, together with a database search, suggests that the NP (I)109(T) mutation would be induced frequently during replication of HPAIVs in brains, but not in lungs. These results demonstrate that the amino acid at position 109 in NP enhances viral RNA synthesis and the pathogenicity of highly pathogenic avian influenza viruses in chickens and that the NP mutation emerges quickly during replication of the viruses in chicken brains.

NP body domain and PB2 contribute to increased virulence of H5N1 highly pathogenic avian influenza viruses in chickens.

The molecular basis of pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in chickens remains largely unknown. H5N1 A/chicken/Yamaguchi/7/2004 virus (CkYM7) replicates rapidly in macrophages and vascular endothelial cells in chickens, causing sudden death without fever or gross lesions, while H5N1 A/duck/Yokohama/aq10/2003 virus (DkYK10) induces high fever, severe gross lesions, and a prolonged time to death, despite the 98% amino acid identity between the two viruses. To explore the molecular basis of this difference in pathogenicity, a series of eight single-gene reassortant viruses from these HPAI viruses were compared for pathogenicity in chickens. Two reassortants possessing the NP or PB2 gene from DkYK10 in the CkYM7 background reduced pathogenicity compared to other reassortants or CkYM7. Inversely, reassortants possessing the NP or PB2 gene of CkYM7 in the DkYK10 background (rgDkYK-PB2(Ck), rgDkYK-NP(Ck)) replicated quickly and reached higher titers than DkYK10, accompanied by more rapid and frequent apoptosis of macrophages. The rgDkYK-NP(Ck) and rgDkYK-PB2(Ck) reassortants also replicated more rapidly in chicken embryo fibroblasts (CEFs) than did rgDkYK10, but replication of these viruses was similar to that of CkYM7 and DkYK10 in duck embryo fibroblasts. A comparison of pathogenicities of seven rgDkYK10 mutants with a single amino acid substitution in NP(Dk) demonstrated that valine at position 105 in the NP(Ck) was responsible for the increased pathogenicity in chickens. NP(Ck), NP(105V), and PB2(Ck) enhanced the polymerase activity of DkYK10 in CEFs. These results indicate that both NP and PB2 contribute to the high pathogenicity of the H5N1 HPAI viruses in chickens, and valine at position 105 of NP may be one of the determinants for adaptation of avian influenza viruses from ducks to chickens.

Phenotypes influencing the transmissibility of highly pathogenic avian influenza viruses in chickens.

To identify critical phenotypes that affect avian influenza virus transmission in chickens, we compared the transmissibility of three H5N1 highly pathogenic viruses of different pathogenicity in chickens by monitoring the exact time of death using wireless thermo-sensors. This study showed that, despite quick deaths, the most virulent H5N1 A/chicken/Yamaguchi/7/2004 transmitted quickly in chickens via contact and airborne routes. Intermediate virulent H5N1 A/chicken/Miyazaki/K11/2007 spread moderately and less virulent H5N1 A/duck/Yokohama/aq10/2003, causing severe clinical signs and a long period to death, spread slowly among the animals. The transmissibility was correlated with virus titres of oropharyngeal and cloacal swabs, and the time for swab virus titres to reach 50 % chicken infective dose affected the transmission speed. These results demonstrate that peak virus titres excreted and the time required for virus titres to reach a minimal chicken infectious dose may be the critical phenotypes influencing the transmissibility of highly pathogenicity avian influenza viruses in chickens.

Association of increased pathogenicity of Asian H5N1 highly pathogenic avian influenza viruses in chickens with highly efficient viral replication accompanied by early destruction of innate immune responses.

The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.