Classifying Ingestive Behavior of Dairy Cows via Automatic Sound Recognition.

Determining ingestive behaviors of dairy cows is critical to evaluate their productivity and health status. The objectives of this research were to (1) develop the relationship between forage species/heights and sound characteristics of three different ingestive behaviors (bites, chews, and chew-bites); (2) comparatively evaluate three deep learning models and optimization strategies for classifying the three behaviors; and (3) examine the ability of deep learning modeling for classifying the three ingestive behaviors under various forage characteristics. The results show that the amplitude and duration of the bite, chew, and chew-bite sounds were mostly larger for tall forages (tall fescue and alfalfa) compared to their counterparts. The long short-term memory network using a filtered dataset with balanced duration and imbalanced audio files offered better performance than its counterparts. The best classification performance was over 0.93, and the best and poorest performance difference was 0.4-0.5 under different forage species and heights. In conclusion, the deep learning technique could classify the dairy cow ingestive behaviors but was unable to differentiate between them under some forage characteristics using acoustic signals. Thus, while the developed tool is useful to support precision dairy cow management, it requires further improvement.

Accurate flexural spring constant calibration of colloid probe cantilevers using scanning laser Doppler vibrometry.

Calibration of the flexural spring constant for atomic force microscope (AFM) colloid probe cantilevers provides significant challenges. The presence of a large attached spherical added mass complicates many of the more common calibration techniques such as reference cantilever, Sader, and added mass. Even the most promising option, AFM thermal calibration, can encounter difficulties during the optical lever sensitivity measurement due to strong adhesion and friction between the sphere and a surface. This may cause buckling of the end of the cantilever and hysteresis in the approach-retract curves resulting in increased uncertainty in the calibration. Most recently, a laser Doppler vibrometry thermal method has been used to accurately calibrate the normal spring constant of a wide variety of tipped and tipless commercial cantilevers. This paper describes a variant of the technique, scanning laser Doppler vibrometry, optimized for colloid probe cantilevers and capable of spring constant calibration uncertainties near ±1%.

Smart Electronic Laboratory Notebooks for the NIST Research Environment.

Laboratory notebooks have been a staple of scientific research for centuries for organizing and documenting ideas and experiments. Modern laboratories are increasingly reliant on electronic data collection and analysis, so it seems inevitable that the digital revolution should come to the ordinary laboratory notebook. The most important aspect of this transition is to make the shift as comfortable and intuitive as possible, so that the creative process that is the hallmark of scientific investigation and engineering achievement is maintained, and ideally enhanced. The smart electronic laboratory notebooks described in this paper represent a paradigm shift from the old pen and paper style notebooks and provide a host of powerful operational and documentation capabilities in an intuitive format that is available anywhere at any time.

Intermittent contact resonance atomic force microscopy.

The intermittent contact resonance atomic force microscopy (ICR-AFM) mode proposed here is a new frequency modulation technique performed in scanning force controlled AFM modes like force volume or peak force tapping. It consists of tracking the change in the resonance frequency of an eigenmode of a driven AFM cantilever during scanning as the AFM probe intermittently contacts a surface at a controlled applied maximum force (setpoint). A high speed data capture was used during individual oscillations to obtain detailed contact stiffness-force curve measurements on a two-phase polystyrene/poly(methyl methacrylate) film with sub-micrometer size domains. Through a suitable normalization, the measurements were analyzed by linear fits to provide an improved quantitative characterization of these materials in terms of their elastic moduli and adhesive properties.

Performance of an image analysis processing system for hen tracking in an environmental preference chamber.

Image processing systems have been widely used in monitoring livestock for many applications, including identification, tracking, behavior analysis, occupancy rates, and activity calculations. The primary goal of this work was to quantify image processing performance when monitoring laying hens by comparing length of stay in each compartment as detected by the image processing system with the actual occurrences registered by human observations. In this work, an image processing system was implemented and evaluated for use in an environmental animal preference chamber to detect hen navigation between 4 compartments of the chamber. One camera was installed above each compartment to produce top-view images of the whole compartment. An ellipse-fitting model was applied to captured images to detect whether the hen was present in a compartment. During a choice-test study, mean ± SD success detection rates of 95.9 ± 2.6% were achieved when considering total duration of compartment occupancy. These results suggest that the image processing system is currently suitable for determining the response measures for assessing environmental choices. Moreover, the image processing system offered a comprehensive analysis of occupancy while substantially reducing data processing time compared with the time-intensive alternative of manual video analysis. The above technique was used to monitor ammonia aversion in the chamber. As a preliminary pilot study, different levels of ammonia were applied to different compartments while hens were allowed to navigate between compartments. Using the automated monitor tool to assess occupancy, a negative trend of compartment occupancy with ammonia level was revealed, though further examination is needed.

Experimental determination of mode correction factors for thermal method spring constant calibration of AFM cantilevers using laser Doppler vibrometry.

Mode correction factors (MCFs) represent a significant adjustment to the spring constant values measured using the thermal cantilever calibration method. Usually, the ideal factor of 0.971 for a tipless rectangular cantilever is used, which adjusts the value by 3% for the first flexural mode. An experimental method for determining MCFs has been developed that relies on measuring the areas under the first few resonance peaks for the flexural mode type. Using this method, it has been shown that MCFs for the first flexural mode of commercially available atomic force microscope cantilevers actually vary from 0.95 to 1.0, depending on the shape and end mass of the cantilever. Triangular shaped cantilevers tend to lower MCFs with tipless versions providing the lowest values. Added masses (including tips) tend to increase the first flexural mode's MCF to higher values with large colloid probes at the high extreme. Using this understanding and applying it to the recently developed laser Doppler vibrometry thermal calibration method it is now possible to achieve very accurate and precise cantilever spring constant calibrations (uncertainties close to ±1%) with commonly available commercial cantilevers such as tipped rectangular and triangular cantilevers, and colloid probes.

Monitoring total endotoxin and (1 --> 3)-beta-D-glucan at the air exhaust of concentrated animal feeding operations.

Mitigation of bioaerosol emissions from concentrated animal feeding operations (CAFOs) demands knowledge of bioaerosol concentrations feeding into an end-of-pipe air treatment process. The aim of this preliminary study was to measure total endotoxin and (1 --> 3)-beta-glucan concentrations at the air exhaust of 18 commercial CAFOs and to examine their variability with animal operation type (swine farrowing, swine gestation, swine weaning, swine finishing, manure belt laying hen, and tom turkey) and season (cold, mild, and hot). The measured airborne concentrations of total endotoxin ranged from 98 to 23,157 endotoxin units (EU)/m3, and the airborne concentrations of total (1 --> 3)-beta-D-glucan ranged from 2.4 to 537.9 ng/m3. Animal operation type in this study had a significant effect on airborne concentrations of total endotoxin and (1 --> 3)-beta-D-glucan but no significant effect on their concentrations in total suspended particulate (TSP). Both endotoxin and (1 --> 3)-beta-D-glucan attained their highest airborne concentrations in visited tom turkey buildings. Comparatively, season had no significant effect on airborne concentrations of total endotoxin or (1 --> 3)-beta-D-glucan. Endotoxin and (1 --> 3)-beta-glucan concentrations in TSP dust appeared to increase as the weather became warmer, and this seasonal effect was significant in swine buildings. Elevated indoor temperatures in the hot season were considered to facilitate the growth and propagation of bacteria and fungi, thus leading to higher biocomponent concentrations in TSP.

Performance of commercial nonmethane hydrocarbon analyzers in monitoring oxygenated volatile organic compounds emitted from animal feeding operations.

Quantifying non-methane hydrocarbons (NMHC) from animal feeding operations (AFOs) is challenging due to the broad spectrum of compounds and the polar nature of the most abundant compounds. The purpose of this study was to determine the performance of commercial NMHC analyzers for measuring volatile organic compounds (VOCs) commonly emitted from AFOs. Three different NMHC analyzers were tested for response to laboratory generated VOCs, and two were tested in the field at a commercial poultry facility. The NMHC analyzers tested included gas chromatography/flame ionization detector (GC/FID), photoacoustic infrared (PA-IR) and photoionization detector (PID). The GC/FID NHHC analyzer was linear in response to nonpolar compounds, but detector response to polar oxygenated compounds were lower than expected due to poor peak shape on the column. The PA-IR NMHC instrument responded well to the calibration standard (propane), methanol, and acetone, but it performed poorly with larger alcohols and ketones and acetonitrile. The PA-IR response varied between compounds in similar compound classes. The PID responded poorly to many of the most abundant VOCs at AFOs, and it underreported alcohols by > 70%. In the field monitoring study, total NMHC concentrations were calculated from sum total of VOC determined using EPA Methods TO-15 and TO-17 with GC-MS compared to results from NMHC analyzers. NMHC GC/FID values were greater than the values calculated from the individual compound measurements. This indicated the presence of small hydrocarbons not measured with TO-15 or TO-17 such as propane. The PA-IR response was variable, but it was always lower than the GC/FID response. Results suggest that improved approaches are needed to accurately determine the VOC profile and NMHC emission rates from AFOs.

Tracking and Characterizing Spatiotemporal and Three-Dimensional Locomotive Behaviors of Individual Broilers in the Three-Point Gait-Scoring System.

Gait scoring is a useful measure for evaluating broiler production efficiency, welfare status, bone quality, and physiology. The research objective was to track and characterize spatiotemporal and three-dimensional locomotive behaviors of individual broilers with known gait scores by jointly using deep-learning algorithms, depth sensing, and image processing. Ross 708 broilers were placed on a platform specifically designed for gait scoring and manually categorized into one of three numerical scores. Normal and depth cameras were installed on the ceiling to capture top-view videos and images. Four birds from each of the three gait-score categories were randomly selected out of 70 total birds scored for video analysis. Bird moving trajectories and 16 locomotive-behavior metrics were extracted and analyzed via the developed deep-learning models. The trained model gained 100% accuracy and 3.62 ± 2.71 mm root-mean-square error for tracking and estimating a key point on the broiler back, indicating precise recognition performance. Broilers with lower gait scores (less difficulty walking) exhibited more obvious lateral body oscillation patterns, moved significantly or numerically faster, and covered more distance in each movement event than those with higher gait scores. In conclusion, the proposed method had acceptable performance for tracking broilers and was found to be a useful tool for characterizing individual broiler gait scores by differentiating between selected spatiotemporal and three-dimensional locomotive behaviors.

Accurate and precise calibration of AFM cantilever spring constants using laser Doppler vibrometry.

Accurate cantilever spring constants are important in atomic force microscopy both in control of sensitive imaging and to provide correct nanomechanical property measurements. Conventional atomic force microscope (AFM) spring constant calibration techniques are usually performed in an AFM. They rely on significant handling and often require touching the cantilever probe tip to a surface to calibrate the optical lever sensitivity of the configuration. This can damage the tip. The thermal calibration technique developed for laser Doppler vibrometry (LDV) can be used to calibrate cantilevers without handling or touching the tip to a surface. Both flexural and torsional spring constants can be measured. Using both Euler-Bernoulli modeling and an SI traceable electrostatic force balance technique as a comparison we demonstrate that the LDV thermal technique is capable of providing rapid calibrations with a combination of ease, accuracy and precision beyond anything previously available.

Atomic Force Microscope Cantilever Flexural Stiffness Calibration: Toward a Standard Traceable Method.

The evolution of the atomic force microscope into a useful tool for measuring mechanical properties of surfaces at the nanoscale has spurred the need for more precise and accurate methods for calibrating the spring constants of test cantilevers. Groups within international standards organizations such as the International Organization for Standardization and the Versailles Project on Advanced Materials and Standards (VAMAS) are conducting studies to determine which methods are best suited for these calibrations and to try to improve the reproducibility and accuracy of these measurements among different laboratories. This paper expands on a recent mini round robin within VAMAS Technical Working Area 29 to measure the spring constant of a single batch of triangular silicon nitride cantilevers sent to three international collaborators. Calibration techniques included reference cantilever, added mass, and two forms of thermal methods. Results are compared to measurements traceable to the International System of Units provided by an electrostatic force balance. A series of guidelines are also discussed for procedures that can improve the running of round robins in atomic force microscopy.

Testing the plastic-wrapped composting system to dispose of swine mortalities during an animal disease outbreak.

Composting has been used to dispose of animal mortalities and infected materials, such as manure and feed, during major animal disease outbreaks. In this study, we adapted the plastic-wrapped mortality composting system developed by the Canadian Food Inspection Agency during the 2004 highly pathogenic avian influenza outbreak to compost swine mortalities. The goals of the study were to evaluate the performance of the plastic-wrapped composting system to dispose of swine mortalities and to field test its ability to eliminate the spread of airborne pathogens through the aeration ducts. Two cover materials, ground cornstalks and woodchips, were tested using passively and actively aerated composting sheds. The mortalities were inoculated with Salmonella spp. and vaccine strains of Bovine herpesvirus-1 and Bovine viral diarrhea virus. Air samples collected from the upper aeration duct (air outlet) during the first 10 d of composting were negative for Salmonella and the viruses tested, which indicated that aerosol transmission of the pathogens was limited. The aeration plenum placed under the mortalities helped to keep conditions aerobic, as O2 concentrations of both passively and actively aerated test units were above 11%. Actively aerated cornstalks had the highest degree-hours (1,462 °C h d-1 ), which was followed by passively aerated cornstalks (1,312 °C h d-1 ), actively aerated woodchips (1,303 °C h d-1 ), and passively aerated woodchips (1,062 °C h d-1 ). After a 7-wk composting period, all three pathogens were inactivated based on quantitative polymerase chain reaction test results. The mortalities were not inoculated with the African swine fever virus, but temperature data showed that if they were, the system had the potential to eliminate this virus.

Perches as Cooling Devices for Reducing Heat Stress in Caged Laying Hens: A Review.

Heat stress is one of the most detrimental environmental challenges affecting the biological process and the related production performance of farm animals, especially in poultry. Commercial laying hens have been bred (selected) for high egg production, resulting in increased sensitivity to heat stress due to breeding-linked metabolic heat production. In addition, laying hens are prone to heat stress due to their inadequate species-specific cooling mechanisms resulting in low heat tolerance. In addition, hens have no sweat glands and feathering covers almost their entire body to minimize body heat loss. The poultry industry and scientists are developing cooling methods to prevent or reduce heat stress-caused damage to chicken health, welfare, and economic losses. We have designed and tested a cooling system using perches, in which chilled water (10 °C) circulates through a conventional perch passing through the layer cages to offer the cooling potential to improve hen health, welfare, and performance during acute and chronic periods of heat stress (35 °C). This review summarizes the outcomes of a multi-year study using the designed cooled perch system. The results indicate that conducting heat from perching hens directly onto the cooled perch system efficiently reduces heat stress and related damage in laying hens. It provides a novel strategy: perches, one key furnishment in cage-free and enriched colony facilities, could be modified as cooling devices to improve thermal comfort for hens during hot seasons, especially in the tropical and subtropical regions.

Microbial assemblages and methanogenesis pathways impact methane production and foaming in manure deep-pit storages.

Foam accumulation in swine manure deep-pits has been linked to explosions and flash fires that pose devastating threats to humans and livestock. It is clear that methane accumulation within these pits is the fuel for the fire; it is not understood what microbial drivers cause the accumulation and stabilization of methane. Here, we conducted a 13-month field study to survey the physical, chemical, and biological changes of pit-manure across 46 farms in Iowa. Our results showed that an increased methane production rate was associated with less digestible feed ingredients, suggesting that diet influences the storage pit's microbiome. Targeted sequencing of the bacterial 16S rRNA and archaeal mcrA genes was used to identify microbial communities' role and influence. We found that microbial communities in foaming and non-foaming manure were significantly different, and that the bacterial communities of foaming manure were more stable than those of non-foaming manure. Foaming manure methanogen communities were enriched with uncharacterized methanogens whose presence strongly correlated with high methane production rates. We also observed strong correlations between feed ration, manure characteristics, and the relative abundance of specific taxa, suggesting that manure foaming is linked to microbial community assemblage driven by efficient free long-chain fatty acid degradation by hydrogenotrophic methanogenesis.

Effects of drying and providing supplemental oxygen to piglets at birth on rectal temperature over the first 24 h after birth.

Neonatal piglets can experience both a decrease in body temperature and hypoxia, increasing risks for pre-weaning mortality. This research evaluated the effects of drying and providing supplemental oxygen to newborn piglets on rectal temperature (RT) over the first 24 h after birth. The study used a CRD with three Intervention Treatments (IT; applied at birth): Control (no intervention), Drying (dried using a desiccant), Oxygen [dried using a desiccant and placed in a chamber (at 40% oxygen concentration) for 20 min]. A total of 42 litters (485 piglets) were randomly allotted to treatments at the start of farrowing. At birth, each piglet was given a numbered ear tag, weighed, and the treatment was applied; RT was measured at 0, 20, 30, 45, 60, 120, and 1440 min after birth. Blood was collected from one piglet from each birth weight quartile within each litter at 24 h after birth to measure plasma immunocrit concentration. There was no effect (P > 0.05) of IT on piglet RT at 0 or 1440 min after birth. Between 20 and 60 min after birth, piglet RT was lower (P ≤ 0.05) for the Control than the Drying treatment, with the Oxygen treatment being intermediate and different (P ≤ 0.05) from the other two IT. The effect of piglet birth weight on responses to IT were evaluated by classifying piglets into Birth Weight Categories (BWC): Light (<1.0 kg), Medium (1.0 to 1.5 kg), or Heavy (>1.5 kg). There were IT by BWC interactions (P ≤ 0.05) for piglet RT at all measurement times between 20 and 120 min after birth. Relative to the Control, the effects of the Drying and Oxygen treatments on RT were greater (P ≤ 0.05) for Light than heavier piglets. Plasma immunocrit concentrations tended (P = 0.07) to be greater for piglets on the Control treatment compared to the other two IT and were lower (P ≤ 0.05) for Light than Heavy piglets, with Medium piglets being intermediate and different (P ≤ 0.05) to the other BWC. In conclusion, drying piglets at birth reduced the extent and duration of RT decline in piglets in the early postnatal period compared to undried piglets, especially for those of low birth weight. However, the combination of drying and placing piglets in an oxygen-rich environment provided no additional benefit over drying alone.

Ammonia emissions from a U.S. broiler house--comparison of concurrent measurements using three different technologies.

There is a need for robust and accurate techniques for the measurement of ammonia (NH3) and other atmospheric pollutant emissions from poultry production facilities. Reasonable estimates of NH3 emission rate (ER) from poultry facilities are needed to guide discussions about the industry's impact on local and regional air quality. The design of these facilities features numerous emission points and results in emission characteristics of relatively low concentrations and exhaust flow rates that vary diurnally, seasonally, and with bird age over a considerable range. These factors combine to render conventional emissions monitoring approaches difficult to apply. Access to these facilities is also often restricted for biosecurity reasons. The three objectives of this study were (1) to compare three methods for measuring exhaust NH3 concentrations and thus ERs, (2) to compare ventilation rates using in situ measured fan characteristics versus using manufacturer sourced fan curves, and (3) to examine limitations of the alternative measurement technologies. In this study, two open-path monitoring systems operating outside of the buildings were compared with a portable monitoring system sampling upstream of a primary exhaust fan. The position of the open-path systems relative to the exhaust fans, measurement strategy adopted, and weather conditions significantly influenced the quality of data collected when compared with the internally located, portable monitoring system. Calculation of exhaust airflow from the facility had a large effect on calculated emissions and assuming that the installed fans performed as per published performance characteristics potentially overestimated emissions by 13.6-26.8%. The open-path measurement systems showed promise for being able to obtain ER measurements with minimal access to the house, although the availability of individual fan characteristics markedly improved the calculated ER accuracy. However, substantial operator skill and experience and favorable weather conditions were required to obtain good quality results.

Pig barns ammonia and greenhouse gas emission mitigation by slurry aeration and acid scrubber.

Livestock production is associated with several gaseous pollutant emissions to the environment. These emissions can degrade local and regional air quality, contribute to surface water eutrophication and acid rain, and contribute to the greenhouse gas footprint of the production sector. Modern production systems must balance animal welfare and environmental pollution potential with economic reality, which is a great challenge to maintain as global demand for animal protein increases. Accordingly, gaseous emission technologies were the main target for this research, in which mitigating gas emissions of ammonia, nitrous oxide, and methane from pig production facilities via slurry aeration system was tested. Five treatments with different airflow rates in the test room were examined continuously over a period of 6 weeks and the results were compared with the control room. Test results indicate that the highest mitigation potentials were 12, 57.6, and 10.4% for nitrous oxide, methane, and ammonia, respectively. Subsequently directing exhaust air into a sulfuric acid air scrubber at 3.0 pH further reduced total ammonia emissions by 80 to 87%.

Wireless 'under the skull' epidural EEG and behavior in piglets during nitrous oxide or carbon dioxide gas euthanasia.

Consciousness is central to animal welfare concerns. Its assessment is most often conducted based on behavior, with a poor understanding of the correspondence between behavior and the neurobiological processes that underlie the subjective experience of consciousness. Recording of brain electrical activity using electrodes placed under the skull improves EEG recording by minimizing artifacts from muscular or cardiac activities, and it can now be combined with wireless recording in free-moving animals. This experiment investigated the correspondence between wireless 'under the skull' epidural EEG and the behavior of 18 five-week-old female piglets undergoing nitrous oxide (N2O) or carbon dioxide (CO2) gradual fill gas euthanasia at 25% replacement rate per minute of the chamber volume. Piglets exposed to CO2 had a peak in EEG total power ('Ptot') during the flailing stage, whereas piglets exposed to N2O had a higher EEG 95% spectral edge frequency ('F95') during their initial explorative behavior phase and a drop in EEG median frequency ('F50') after loss of posture. Loss of posture without righting attempt, as the last behavioral state observed during euthanasia, preceded the onset of transitional EEG on average by 0.9 and 3.1 min (for CO2 and N2O treatments, respectively), and the onset of isoelectric EEG by 4.5 and 6.2 min (for CO2 and N2O treatments, respectively). Paddling movements occurred shortly before and during transitional EEG but never during isoelectric EEG, whereas gasps persisted after the EEG had become isoelectric. The dynamics of EEG spectral changes were complex to interpret in relation to the degree of consciousness, but isoelectric EEG as an unequivocal indicator of unconsciousness appeared several minutes after loss of posture with no righting attempt. This leaves a window of uncertainty in regards to the potential for consciousness after loss of posture during gradual fill gas euthanasia in piglets.

Factors Affecting Trailer Thermal Environment Experienced by Market Pigs Transported in the US.

Extreme weather conditions challenge pig thermoregulation during transport and are addressed by the National Pork Board (NPB) Transport Quality Assurance® (TQA) program that provides guidelines for trailer boarding, bedding, and misting. These guidelines are widely applied, yet very little is known about the microenvironment within the trailer. In this study, TQA guidelines (V4) were evaluated via extensive thermal environment measurements during transport in order to evaluate spatial variability and implications on ventilation pattern. Effects of trailer management strategies including bedding, boarding, and misting were examined and the trailer was monitored for interior temperature rise and THI responses within six separate zones. The trailer thermal environment was not uniformly distributed in the colder trips with the top front and bottom zones were the warmest, indicating these zones had the majority of outlet openings and experienced air with accumulated sensible and latent heat of the pigs. Relatively enhanced thermal environment uniformity was observed during hot trips, suggesting that ventilation patterns and ventilation rate were different for colder vs. warmer weather conditions. Misting applied prior to transport cooled interior air temperature, but also created high THI conditions in some cases. Neither boarding and bedding combinations in the TQA nor boarding position showed impacts on trailer interior temperature rise or spatial distribution of temperature inside the trailer.

A Two-Step Process of Nitrous Oxide before Carbon Dioxide for Humanely Euthanizing Piglets: On-Farm Trials.

Current methods of euthanizing piglets are raising animal welfare concerns. Our experiment used a novel two-step euthanasia method, using nitrous oxide (N2O) for six minutes and then carbon dioxide (CO2) on compromised 0- to 7-day-old piglets. A commercial euthanasia chamber was modified to deliver two euthanasia treatments: the two-step method using N2O then CO2 (N2O treatment) or only CO2 (CO2 treatment). In Experiment 1, 18 piglets were individually euthanized. In Experiment 2, 18 groups of four to six piglets were euthanized. In the N2O treatment, piglets lost posture, indicating the onset of losing consciousness, before going into CO2 where they showed heavy breathing and open-mouth breathing; whereas piglets in the CO2 treatment did not lose posture until after exhibiting these behaviors (p ≤ 0.004). However, piglets in the N2O treatment took longer to lose posture compared to the CO2 treatment (p < 0.001). Piglets in the N2O treatment displayed more behavioral signs of stress and aversion: squeals/minute (p = 0.004), escape attempts per pig (p = 0.021), and righting responses per pig (p = 0.084) in a group setting. In these regards, it cannot be concluded that euthanizing piglets for 6 min with N2O and then CO2 is more humane than euthanizing with CO2 alone.