Development of an apparatus for evaluating the visual and olfactory discrimination ability of cattle under controlled target visibility.

Ungulates visually and olfactorily discriminating between vegetation patches in grasslands often encounter restriction of target visibility due to light intensity changes; however, little is known about their performance in such a context. We developed and tested an apparatus for evaluating the visual and olfactory discrimination ability of cattle under controlled target visibility, focusing on the discrimination at a short distance. The apparatus was designed to contain a discrimination target under a sliding cover of variable light transmission levels and behind a vent of a fixed size and aperture so as to control the visibility of the target (14-100% restrictions) while ensuring a constant level of odor. Twelve Japanese Black cows were allowed to choose between two apparatuses presenting a pair of targets: green forage versus empty, green forage versus dead forage, or green forage versus green-dead mixture. Cows rapidly learned to slide open the cover to reach the selected target, consistently chose the green forage against the alternative except against the green-dead mixture under 100% visual restriction, and remembered the reaching procedure for at least 16 days. The results indicate the usefulness of the apparatus for assessing close visual and olfactory discrimination ability of cattle in detail.

Interleukin-17A Peptide Aptamers with an Unexpected Binding Moiety Selected by cDNA Display under Heterogenous Conditions.

Peptide-based drugs are an attractive new modality of therapeutics, and in vitro selection from a large-scale library is a powerful way to identify new lead sequences. In conventional screenings, peptide specificity and stability in physiological heterogenous environments are not evaluated, which sometimes makes subsequent optimization difficult. Here we show that selection using a cDNA display system can be performed in a high percentage of serum and that this might be an option to select molecules with high potency and stability in a biological context. Specifically, we chose interleukin-17A as a target protein and performed in vitro selection of cyclic peptide aptamers from a library of approximately 1012 members in the presence of serum. The selected molecules had nanomolar affinity to the target and were stable in serum. Interestingly, we found that a component of the DNA linker that connected the peptide and cDNA may play a pivotal role in target binding.

Effects of Heat Stress on Heart Rate Variability in Free-Moving Sheep and Goats Assessed With Correction for Physical Activity.

Heart rate variability (HRV) is the heart beat-to-beat variation under control of the cardiovascular function of animals. Under stressed conditions, cardiac activity is generally regulated with an upregulated sympathetic tone and withdrawal of vagal tone; thus, HRV monitoring can be a non-invasive technique to assess stress level in animals especially related to animal welfare. Among several stress-induced factors, heat stress is one of the most serious causes of physiological damage to animals. The aim of this study was to assess the effects of heat stress on HRV in small ruminants under free-moving conditions. In three experimental periods (June, August, and October), inter-beat intervals in sheep and goats (three for each) in two consecutive days were measured. HRV parameters were calculated from the inter-beat interval data by three types of analyses: time domain, frequency domain, and non-linear analyses. The temperature-humidity index (THI) was used as an indicator of heat stress, and vectorial dynamic body acceleration (VeDBA) was calculated to quantify the physical activity of the animals tested. First, we investigated correlations of THI and VeDBA with HRV parameters; subsequently, THI was divided into five categories according to the values obtained (≤ 65, 65-70, 70-75, 75-80, and >80), and the effects of the THI categories on HRV parameters were investigated with and without correcting for the effects of physical activity based on the VeDBA. The results indicated that HRV significantly decreased with increasing THI and VeDBA. For non-linear HRV parameters that were corrected for the effects of physical activity, it was suggested that there would be a threshold of THI around 80 that strongly affected HRV; high heat stress can affect the autonomic balance of animals non-linearly by inducing the sympathetic nervous system. In conclusion, to assess psychophysiological conditions of unrestrained animals by HRV analysis, the confounding effect of physical activity on HRV should be minimized for a more precise interpretation of the results.

Selection of Peptides that Associate with Dye-Conjugated Solid Surfaces in a pH-Dependent Manner Using cDNA Display.

Peptides that recognize artificial materials including synthetic polymers and small molecules are drawing attention in the fields of biotechnology and chemical biology. In particular, reversible peptide aptamers that associate with the target molecules only under specific conditions are interesting. In this work, peptide aptamers that recognize a phenolphthalein derivative (PhP: a pH-sensitive organic dye) immobilized on a solid surface in a pH-dependent manner were selected using an in vitro display method (cDNA display). Considering the hydrophobic and aromatic nature of PhP, we prepared a biased DNA library (3A library) that encodes more aromatic amino acids than the standard random codon and performed seven rounds of selection from >1010 peptide species. The selected peptides including LVFLIWWM (LV59) associated with PhP-modified solid support (sepharose resin and magnetic beads) in neutral buffer but readily dissociated under basic conditions where PhP undergoes large structural change from lactone to quinoid, which is accompanied by increase of hydrophilicity and anionic charge. Control experiments suggested that LV59 recognized both phenol and lactone moieties, and the association under neutral pH is mainly driven by π-stacking and hydrophobic interaction between the peptide and PhP. Notably, however, total hydrophobicity and number of aromatic rings did not completely explain the affinity, and sequence specificity was observed to some extent. After further optimization, this interaction pair would be practically useful for protein purification.

Can cattle visually discriminate between green and dead forages at a short distance while moving in the field?

Relatively little is known about the ability of ungulates to visually discriminate vegetation patches while foraging on grasslands despite extensive studies with man-made stimuli presented indoors. This study aimed to assess visual discrimination ability of cattle (Bos taurus) under conditions closer to the actual foraging situation. Twelve Japanese Black cows were afforded four successive opportunities to choose between green and dead forages presented as 25 × 25 cm patches 1, 2, or 3 m ahead while walking through a 25-m-long field area. Apparatuses for presenting the forages as visual stimuli were designed to minimize olfactory cues. The green forage differed from the dead forage in appearance (color and texture) and quality (digestible dry matter and crude protein). Cows preferred the green forage to the dead forage and were able to use the forages as visual cues to discriminate them. The proportion of green forage choices was 0.70-0.72 (different from the chance at P < 0.001), 0.57 (P < 0.05), and 0.53 (P ≥ 0.1) at the distances of 1, 2, and 3 m, respectively. The results indicate that the ability of ungulates to visually discriminate vegetation patches during foraging in grasslands would not be as high as that expected from the visual acuity reported in the previous indoor studies.

A novel immuno-PCR method using cDNA display.

Immuno-PCR (IPCR) provides sensitive and versatile detection of a variety of antigens by conjugating a PCR-amplifiable DNA reporter to a specific antibody or an aptamer. Several methodologies have been developed to prepare appropriate DNA-antibody conjugates, but in most cases, it remains difficult to label polypeptides with high site-specificity and fixed stoichiometry. To address this issue, we first demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA via puromycin at the single molecule level. Several other in vitro display technologies (e.g., ribosome display, mRNA display) have similar simple nucleic acid-peptide linkage. However, they should be unsuitable for diagnostic applications because of their lability against heat and RNase. The newly developed system here, termed cDNA display mediated immuno-PCR (cD-IPCR), proved to work in direct- and sandwich-type detection of target proteins. Detection of a target in serum was also possible, using a VHH (variable domain of the heavy chain of a heavy chain antibody) antibody as a binding molecule. Although further improvement on sensitivity and quantitativity is necessary before the method becomes useful, we believe this work demonstrated a potential of cD-IPCR as an alternative novel format of IPCR.

Correcting the Activity-Specific Component of Heart Rate Variability Using Dynamic Body Acceleration Under Free-Moving Conditions.

Heart rate variability (HRV) analysis is a widely used technique to assess sympatho-vagal regulation in response to various internal or external stressors. However, HRV measurements under free-moving conditions are highly susceptible to subjects' physical activity levels because physical activity alters energy metabolism, which inevitably modulates the cardiorespiratory system and thereby changes the sympatho-vagal balance, regardless of stressors. Thus, researchers must simultaneously quantify the effect of physical activity on HRV to reliably assess sympatho-vagal balance under free-moving conditions. In the present study, dynamic body acceleration (DBA), which was developed in the field of animal ecology as a quantitative proxy for activity-specific energy expenditure, was used as a factor to correct for physical activity when evaluating HRV in freely moving subjects. Body acceleration and heart inter-beat intervals were simultaneously measured in cattle and sheep, and the vectorial DBA and HRV parameters were evaluated at 5-min intervals. Next, the effects of DBA on the HRV parameters were statistically analyzed. The heart rate (HR) and most of the HRV parameters were affected by DBA in both animal species, and the inclusion of the effect of DBA in the HRV analysis greatly influenced the frequency domain and nonlinear HRV parameters. By removing the effect of physical activity quantified using DBA, we could fairly compare the stress levels of animals with different physical activity levels under different management conditions. Moreover, we analyzed and compared the HRV parameters before and after correcting for the mean HR, with and without inclusion of DBA. The results were somewhat unexpected, as the effect of DBA was a highly significant source of HRV also in parameters corrected for mean HR. In conclusion, the inclusion of DBA as a physical activity index is a simple and useful method for correcting the activity-specific component of HRV under free-moving conditions.

Interspecific comparison of allometry between body weight and chest girth in domestic bovids.

The sizes of body parts often co-vary through exponential scaling, known as allometry. The evolution of allometry is central to the generation of morphological diversity. To make inferences regarding the evolved responses in allometry to natural and artificial selection, we compared allometric parameters (slope and intercept) among seven species and breeds of domestic bovids using cross-sectional ontogenetic data and attempted to interpret the differences in these parameters. The allometric slopes were not different among some species, whereas those between breeds within species were, indicating that the slopes were typically invariant but could be changed under strong, specific selection. With the exception of yak, the differences in the intercept independent of the slopes (the alternative intercept) among species might better correspond to their divergence times than the differences in allometric slope, and the remarkably higher alternative intercept found in yaks can be explained by their unique morphological evolution. These findings provide evidence that differences in the alternative intercept can retain traces of the phylogenetic changes derived from differentiation and evolution.

Health and mineral nutrition status of yaks in southern Mustang, Nepal.

Biochemical values and mineral concentrations in blood plasma were investigated to evaluate the statuses of health and mineral nutrition among yaks in Mustang District, Nepal. In total, 118 plasma samples of female yaks collected in April and September/October of 2013-2015 were offered. Seventy-four percent of yaks showed lower plasma total-cholesterol concentrations than the lowest limit of reference range (100 mg/dL) and the values in spring (83.41 mg/dL) were lower (P < 0.05) than those in autumn (95.05 mg/dL). All the yaks had lower plasma albumin concentrations than the lowest limit of reference range (3.0 g/dL) and 66% of yaks showed lower plasma inorganic phosphorus concentrations than the critical level of phosphorus deficiency (4.5 mg/dL). Thirty-five percent of yaks showed lower plasma calcium concentrations than the lowest limit of normal range (8 mg/dL) and the concentrations were lower in spring than in autumn (P < 0.01). Seventy-five percent of yaks presented lower copper concentrations than the critical level (0.65 mg/L) and the concentrations were lower in spring than in autumn (P < 0.01). Since the low plasma total-cholesterol might have indicated shortage of dry matter and energy intake, attention should be paid to the nutritional statuses of energy, phosphorus, calcium and copper in winter and early spring.

Estimation of potassium and magnesium flows in animal production in Dianchi Lake basin, China.

The objectives of this study were to estimate and evaluate potassium (K) and magnesium (Mg) budgets and flows of animal production in the basin of Dianchi Lake, China. Feed sampling and farmer interviews were conducted in field surveys. The supplies of K and Mg from local and external feeds and the retention, production and excretion of animals were calculated individually for dairy cows, fattening pigs, breeding sows, and broilers and laying hens. The K and Mg flows on a regional level were estimated using the individual budgets. At the individual level, in dairy cattle, the K and Mg supplied from local feeds accounted for large parts of the total nutrient intakes, whereas in the other animal categories most of the K and Mg in the feeds depended on external resources. Our findings also suggested that excessive Mg intake resulted in high Mg excretion and low use efficiency in dairy cattle and fattening pigs. At the regional level, the K and Mg amounts of manure produced and applied in the area (K: 339 and Mg: 143 t/year) exceeded those used as local feeds. Our results imply the animal production potentially increased the K and Mg loads in the regional agriculture system.

Estimation of nitrogen and phosphorus flows in livestock production in Dianchi Lake basin, China.

We assessed the nitrogen (N) and phosphorus (P) flows in intensified livestock production systems by investigating nutrient budgets and cycling in the basin of Dianchi Lake, one of the most eutrophic lakes in China. We conducted field surveys based on feed samplings and interviews of livestock farmers. The N and P in local and external feeds, animal body retentions, animal products and excretions were calculated at the individual level for dairy cattle, fattening pigs, breeding sows, broilers and laying hens. The N and P flows in the total livestock production system in the area were estimated by multiplying the individual N and P budgets by the number of animals. For the dairy and fattening pig productions, N and P supplied from local crops or by-products accounted for large parts of the inputs. For the other livestock categories, most of the N and P inputs depended on external resources. The N and P outputs through animal manure into the cropland were 287 and 66 kg/ha/year, respectively, which were higher than the N and P inputs into the livestock production systems from the cropland. The N and P loads from manure should be reduced for the establishment of sustainable agricultural production systems.

Application of overall dynamic body acceleration as a proxy for estimating the energy expenditure of grazing farm animals: relationship with heart rate.

Estimating the energy expenditure of farm animals at pasture is important for efficient animal management. In recent years, an alternative technique for estimating energy expenditure by measuring body acceleration has been widely performed in wildlife and human studies, but the availability of the technique in farm animals has not yet been examined. In the present study, we tested the potential use of an acceleration index, overall dynamic body acceleration (ODBA), as a new proxy for estimating the energy expenditure of grazing farm animals (cattle, goats and sheep) at pasture with the simultaneous evaluation of a conventional proxy, heart rate. Body accelerations in three axes and heart rate for cows (n = 8, two breeds), goats (n = 6) and sheep (n = 5) were recorded, and the effect of ODBA calculated from the body accelerations on heart rate was analyzed. In addition, the effects of the two other activity indices, the number of steps and vectorial dynamic body acceleration (VeDBA), on heart rate were also investigated. The results of the comparison among three activity indices indicated that ODBA was the best predictor for heart rate. Although the relationship between ODBA and heart rate was different between the groups of species and breeds and between individuals (P<0.01), the difference could be explained by different body weights; a common equation could be established by correcting the body weights (M: kg): heart rate (beats/min) = 147.263∙M-0.141 + 889.640∙M-0.179∙ODBA (g). Combining this equation with the previously reported energy expenditure per heartbeat, we estimated the energy expenditure of the tested animals, and the results indicated that ODBA is a good proxy for estimating the energy expenditure of grazing farm animals across species and breeds. The utility and simplicity of the procedure with acceleration loggers could make the accelerometry technique a worthwhile option in field research and commercial farm use.