Comparison of immunoassay and LC-tandem mass spectrometry analyses of ractopamine in hog oral fluid.

The accurate detection of ractopamine in food animals is crucial for marketing since some entities require animals or animal carcasses to be free of ractopamine residues. Field-based ractopamine screening tests that are rapid, sensitive, and capable of high-throughput are highly desirable to ensure that inadvertent exposure to ractopamine did not occur in animals marketed as animals that have not been fed ractopamine. An immunochemically based lateral flow assay was used to analyze oral fluids from hogs never exposed to ractopamine and from hogs that were presumed positives and results were confirmed using an enhanced sensitivity LC-MSMS method. We found that an immunochemically based lateral flow system having a working range of 2.5 to 15 ng mL-1 worked well as a screening assay with 1.7% false positive results in freshly collected hog oral fluids. Using ractopamine glucuronide standards and LC-MSMS, we determined that the false positive results were not due to the presence of ractopamine glucuronide metabolites in oral fluids.

Are infrared thermography, feeding behavior, and heart rate variability measures capable of characterizing group-housed sow social hierarchies?

Group gestation housing is quickly becoming standard practice in commercial swine production. However, poor performance and welfare in group housed sows may result from the formation and maintenance of the social hierarchy within the pen. In the future, the ability to quickly characterize the social hierarchy via precision technologies could be beneficial to producers for identifying animals at risk of poor welfare outcomes. Therefore, the objective of this study was to investigate the use of infrared thermography (IRT), automated electronic sow feeding systems, and heart rate monitors as potential technologies for detecting the social hierarchy within five groups of sows. Behavioral data collection occurred for 12 h after introducing five sow groups (1-5; n = 14, 12, 15, 15, and 17, respectively) to group gestation housing to determine the social hierarchy and allocate individual sows to 1 of 4 rank quartiles (RQ 1-4). Sows within RQ1 were ranked highest while RQ4 sows were ranked lowest within the hierarchy. Infrared thermal images were taken behind the neck at the base of the ear of each sow on days 3, 15, 30, 45, 60, 75, 90, and 105 of the experiment. Two electronic sow feeders tracked feeding behavior throughout the gestation period. Heart rate monitors were worn by 10 randomly selected sows per repetition for 1 h prior to and 4 h after reintroduction to group gestation housing to collect heart rate variability (HRV). No differences were found between RQ for any IRT characteristic. Sows within RQ3 and RQ4 had the greatest number of visits to the electronic sow feeders overall (P < 0.04) but spent shorter time per visit in feeders (P < 0.05) than RQ1 and RQ2 sows. There was an interaction of RQ with hour for feed offered (P = 0.0003), with differences between RQ occurring in hour 0, 1, 2, and 8. Higher-ranked sows (RQ1 and RQ2) occupied the feeder for longer during the first hour than lower ranking sows (RQ3 and RQ4; P < 0.04), while RQ3 sows occupied the feeder longer than RQ1 sows during hour 6, 7, and 8 (P < 0.02). Heart beat interval (RR) collected prior to group housing introduction differed between RQ (P < 0.02 for all), with RQ3 sows exhibiting the lowest RR, followed by RQ4, RQ1, and RQ2. Rank quartile also affected standard deviation of RR (P = 0.0043), with RQ4 sows having the lowest, followed by RQ1, RQ3, and RQ2 sows. Overall, these results indicate that feeding behavior and HRV measures may be capable of characterizing social hierarchy in a group housing system.

Sows that are housed in groups establish a social hierarchy to gain preferential access to needed resources, such as feed. The establishment and maintenance of this social hierarchy may lead to reduced welfare and performance for certain sows. Therefore, the purpose of this study was to determine whether infrared thermography, feeding behavior, and heart rate variability measurement could identify the social hierarchy. In the future, the ability to automatically detect the social hierarchy within group housed pens using technology may give a producer the ability to mitigate any hierarchy-related welfare and performance issues on their own farm. Our results show that feeding behavior collected by an automated feeding system may be a promising tool for future social hierarchy detection. Additionally, measures related to changes in heart rate over time are capable of identifying high- and low-ranked sows when the measures are collected before sows are moved into groups. Therefore, technologies capable of measuring feeding behavior and changes in heart rate over time may be promising for future automated detection of the sow social hierarchy.

Characterizing the effect of incrementally increasing dry bulb temperature on linear and nonlinear measures of heart rate variability in nonpregnant, mid-gestation, and late-gestation sows.

Characterizing the sow physiological response to an increased heat load is essential for effective heat stress mitigation. The study objective was to characterize the effects of a 400-min heating episode on sow heart rate variability (HRV) at different reproductive stages. HRV is a commonly used noninvasive proxy measure of autonomic function. Twenty-seven sows were enrolled in the study according to their gestation stage at time of selection: 1) nonpregnant (NP; n = 7), 2) mid-gestation (MID; 57.3 ± 11.8 d gestation; n = 11), and 3) late-gestation (LATE; 98.8 ± 4.9 d gestation; n = 8). The HRV data utilized in the study were collected from each pig as the dry bulb temperature in the room increased incrementally from 19.84 ± 2.15 °C to 35.54 ± 0.43 °C (range: 17.1-37.5 °C) over a 400-min period. After data collection, one 5-min set of continuous heart rate data were identified per pig for each of nine temperature intervals (19-20.99, 21-22.99, 23-24.99, 25-26.99, 27-28.99, 29-30.99, 31-32.99, 33-34.99, and 35-36.99 °C). Mean inter-beat interval length (RR), standard deviation of r-r intervals (SDNN), root mean square of successive differences (RMSSD), high frequency spectral power (HF), sample entropy (SampEn), short-term detrended fluctuation analysis (DFAα1), and three measures (%REC, DET, LMEAN) derived from recurrence quantification analysis were calculated for each data set. All data were analyzed using the PROC GLIMMIX procedure in SAS 9.4. Overall, LATE sows exhibited lower RR than NP sows (P < 0.01). The standard deviation of r-r intervals and RMSSD differed between each group (P < 0.01), with LATE sows exhibiting the lowest SDNN and RMSSD and NP sows exhibiting the greatest SDNN and RMSSD. Late-gestation sows exhibited lower HF than both MID and NP sows (P < 0.0001), greater DFA values than NP sows (P = 0.05), and greater DET compared to MID sows (P = 0.001). Late-gestation also sows exhibited greater %REC and LMEAN compared to MID (P < 0.01) and NP sows (all P < 0.01). In conclusion, LATE sows exhibited indicators of greater autonomic stress throughout the heating period compared to MID and NP sows. However, temperature by treatment interactions were not detected as dry bulb increased. Future studies are needed to fully elucidate the effect of gestational stage and increasing dry bulb temperature on sow HRV.

Pregnant pigs may be at a higher risk of poor physiological outcomes due to heat exposure compared to mature female pigs that are not pregnant. The purpose of this study was to evaluate the stress response of pregnant pigs to increasing environmental temperatures using heart rate variability, a noninvasive measure commonly used to evaluate the physiological stress response. Our findings show that pregnant pigs, particularly those who are closer to giving birth, exhibit greater evidence of physiological stress compared to pigs who are not pregnant. However, we did not find evidence that increasing environmental temperature throughout the experimental period was a primary reason for the increased stress exhibited by pregnant pigs. It is possible that the physiological changes that normally occur during pregnancy may have masked the physiological stress response typically associated with increased heat exposure.

Evaluation of rapid and standard tandem mass spectrometric methods to analyse veterinary drugs and their metabolites in antemortem bodily fluids from food animals.

Antemortem bodily fluids can serve as an indicator of veterinary medicine exposure prior to food animal slaughter. A multi-residue, rapid screen electrospray ionisation mass spectrometric (RS-ESI-MS) method was developed to analyse 10 veterinary drugs or metabolites (clenbuterol, erythromycin, flunixin, 5-hydroxyflunixin, meloxicam, ractopamine, ractopamine-glucuronide, salbutamol, tylosin, and zilpaterol) in hog oral fluid and bovine urine. Simple acetonitrile extraction with salting-out was employed to remove the analytes from matrices in less than 30 minutes. Instrumental analysis time was < 1 min/injection. Regression coefficients of matrix-matched calibration curves ranged 0.9743-0.9999 across all compounds with limits of detection ranging from 0.46-108 ng mL-1 for cattle urine and 0.19-64.4 ng mL-1 for hog oral fluid across all analytes. Except for ractopamine-glucuronide, analyte recoveries ranged from 92.7-106% for oral fluid and urine fortified at 30, 100, and 300 ng mL-1, with inter-day variations of < 25%. Ractopamine-glucuronide recovery was 93.3% for oral fluid fortified at 300 ng mL-1. The RS-ESI-MS method accurately identified ractopamine and/or ractopamine-glucuronide in incurred cattle urine with results correlating well with traditional LC-MS/MS and HPLC fluorescence methods. As far as we are aware, this is the first report of the direct quantification of ractopamine-glucuronide from biological matrices without lengthy hydrolysis and cleanup steps.

Characterizing the postnatal hypothalamic-pituitary-adrenal axis response of in utero heat stressed pigs at 10 and 15 weeks of age.

In utero heat stress alters postnatal physiological and behavioral stress responses in pigs. However, the mechanisms underlying these alterations have not been determined. The study objective was to characterize the postnatal hypothalamic-pituitary-adrenal axis response of in utero heat-stressed pigs. Pigs were subjected to a dexamethasone suppression test followed by a corticotrophin releasing hormone challenge at 10 and 15 weeks of age. Following the challenge, hypothalamic, pituitary, and adrenal tissues were collected from all pigs for mRNA abundance analyses. At 10 weeks of age, in utero heat-stressed pigs had a reduced (P < 0.05) cortisol response to the corticotrophin releasing hormone challenge versus controls. Additionally, the cortisol response tended to be greater overall (P < 0.10) in 15 versus 10-week-old pigs in response to the dexamethasone suppression test. The cortisol response tended to be reduced overall (P < 0.10) in 15 versus 10-week-old pigs in response to the corticotrophin releasing hormone challenge. Hypothalamic corticotropin releasing hormone mRNA abundance tended to be greater (P < 0.10) in in utero heat-stressed versus control pigs at 15-weeks of age. In summary, in utero heat stress altered some aspects of the hypothalamic-pituitary-adrenal axis related to corticotropin releasing hormone signaling, and age influenced this response.

The Effect of Demonstrator Social Rank on the Attentiveness and Motivation of Pigs to Positively Interact with Their Human Caretakers.

In this study, we addressed the social attentiveness, as well as the phenomenon of social facilitation and inhibition in the context of a positive human-pig relationship. Specifically, we investigated whether the social rank of an experienced pig (termed "demonstrator") has an effect on the attentiveness of the remaining pen mates (N = 40) when they observe the demonstrator being gently handled by a stockperson from behind an acrylic panel. We found that pigs preferentially attended to dominant demonstrators rather than subordinate demonstrators during their gentle handling sessions with the stockperson. Additionally, we also examined whether the presence of a demonstrator pig of different social rank, who previously established a positive relationship with the stockperson in presence of conspecifics, affects the behavior and motivation of their pen mates to positively interact with the stockperson. To test for the effect of the presence and demonstrator's social rank on pen mate interactions with the stockperson, we evaluated the behavior of domestic pigs (N = 65) toward the stockperson using a human-approach test in their home-pen. Pigs showed a decrease in their motivation to positively interact with the stockperson when a socially dominant demonstrator was present, behaving similarly to animals receiving minimal human contact (control group). Overall, they exhibited a greater latency to physical contact, a lower acceptance of stroking, and spent more time looking at the stockperson compared to pigs exposed to subordinate demonstrators. Taken together, these findings expand our current understanding of pigs' cognition and social behavior, and the nature of social attention bias in farm animals. Our findings indicate that positive handling of previously selected subordinate demonstrators seems to be the best strategy to reduce the level of fear in large groups of pigs.

Pharmacokinetics of oral firocoxib in un-weaned calves.

The objective of this study was to determine the pharmacokinetics of firocoxib after oral administration in un-weaned calves. Eight Holstein calves with a mean age of 36 days and a mean weight of 55 kg were administered a single oral dose of 227 mg firocoxib. The resulting mean dosage was 4.2 mg/kg (range 3.5-5.0 mg/kg). Blood was collected prior to drug administration and at 2, 4, 6, 8, 24, 48, 72, and 96 h after treatment. Firocoxib concentrations in plasma were determined using liquid chromatography-tandem mass spectrometry. Using computer software, pharmacokinetic parameters were found to fit best with a one-compartment model. Mean Cmax was 0.9 µg/ml (range 0.570-1.254), and Tmax was estimated to be 7 h (range 4-8 h). The estimated T1/2 was 15.3 h. The pharmacokinetics of firocoxib after oral dosing are similar to those in dogs, with the exception of a T1/2 that is approximately twice as long. Based on the similar pharmacokinetics, it is possible that a dose of 227 mg firocoxib orally could provide an analgesic effect in un-weaned calves.

Do Domestic Pigs Acquire a Positive Perception of Humans through Observational Social Learning?

Farm animals can perceive humans positively by observing another animal being positively handled. This study evaluated whether pigs acquire a positive perception of humans after observing either a high or low socially ranked conspecific receiving gentle handling. Seventy-five 21-week-old pigs were housed in 15 nursery pens (five pigs/pen) and randomly assigned to one of three pen treatments: Dominant Demonstrator Group (DDG), Subordinate Demonstrator Group (SDG) and Control Group (CG). Pigs from DDG and SDG observed a high and low socially ranked conspecific ("demonstrator"), respectively, while the demonstrator received gentle stroking and a sucrose solution for 10 min, twice a day for 5 weeks. Control group pigs received minimal human contact. Following treatment, the behavior and heart rate variability of non-demonstrator pigs were evaluated in response to a stockperson in an open-field test. Pigs from the DDG and SDG contacted the stockperson sooner (p < 0.001), spent more time investigating the stockperson (p < 0.05), accepted more stroking (p < 0.001) and exhibited a lower low/high frequency ratio (p = 0.015) compared to the CG. No differences in learning between the pigs from the DDG and SDG were found. These results suggest that pigs can learn to perceive humans positively through observational social learning, regardless of the demonstrator conspecific's social rank.

In utero heat stress alters the postnatal innate immune response of pigs.

The effects of in utero heat stress (IUHS) range from decreased growth performance to altered behavior, but the long-term impact of IUHS on postnatal innate immune function in pigs is unknown. Therefore, the study objective was to determine the effects of early gestation IUHS on the immune, metabolic, and stress response of pigs subjected to an 8 hr lipopolysaccharide (LPS) challenge during postnatal life. Twenty-four pregnant gilts were exposed to thermoneutral (TN; n = 12; 17.5 ± 2.1 °C) or heat stress (HS; n = 12; cyclic 26 to 36 °C) conditions from days 6 to 59 of gestation, and then TN conditions (20.9 ± 2.3 °C) from day 60 of gestation to farrowing. At 12 wk of age, 16 IUHS and 16 in utero thermoneutral (IUTN) pigs were selected, balanced by sex and given an intravenous injection of LPS (2 µg/kg BW mixed with sterile saline [SAL] and injected at 2 µL/kg BW) or SAL (2 µL/kg BW). Body temperature was monitored every 30 min, and blood was obtained at 0, 1, 2, 3, 4, 6, and 8 hr following the LPS challenge. Blood samples were analyzed for glucose, insulin, non-esterified fatty acids (NEFA), cortisol, and cytokine concentrations. In addition, white blood cell counts were determined at 0 and 4 hr. Hour 0 data were used as covariates. Body temperature was increased (P < 0.01) in LPS (40.88 ± 0.08 °C) vs. SAL (39.83 ± 0.08 °C) pigs. Eosinophils tended to be decreased overall (P = 0.09; 43.9%) in IUHS vs. IUTN pigs. Glucose concentrations were reduced overall (P = 0.05; 5.9%) in IUHS vs. IUTN pigs. The NEFA concentrations tended to be greater (P = 0.07; 143.4%) in IUHS-LPS pigs compared with all other treatments, and IUTN-LPS pigs tended to have greater (127.4%) circulating NEFA concentrations compared with IUTN-SAL and IUHS-SAL pigs. Cortisol was increased (P = 0.04) in IUHS-LPS compared with IUTN-LPS pigs at 3 hr (21.5%) and 4 hr (64.3%). At 1 hr, tumor necrosis factor α was increased (P = 0.01; 115.1%) in IUHS-LPS compared with IUTN-LPS pigs. Overall, interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were greater (P < 0.04; 281.3% and 297.8%, respectively) in IUHS-LPS pigs compared with all other treatments, and IUTN-LPS pigs had increased IL-1ß and IL-6 concentrations compared with IUTN-SAL and IUHS-SAL pigs. In summary, IUHS altered the postnatal cytokine, metabolic, and physiological stress response of pigs during postnatal life, which may have negative implications toward the innate immune response of IUHS pigs to pathogens.

Evaluation and mitigation of the effects of in utero heat stress on piglet growth performance, postabsorptive metabolism, and stress response following weaning and transport.

In utero heat stress (IUHS) increases the energy requirements of pigs during postnatal life, and this may compound weaning and transport stress. The study objective was to evaluate and mitigate the negative effects of IUHS following weaning and transport through the provision of a nutrient-dense (ND) nursery diet formulated to meet the greater energy requirements of IUHS pigs during the first 14 d postweaning and transport. Twenty-four pregnant gilts were exposed to thermoneutral (TN; n = 12; 17.5 ± 2.1 °C) or heat stress (HS; n = 12; cycling 26 to 36 °C) conditions for the first half of gestation (day 6 to 59) and then TN conditions (20.9 ± 2.3 °C) until farrowing. Nine TN gilts and 12 HS gilts produced litters. At weaning (16.2 ± 0.4 d), mixed-sex piglets (N = 160; 4.78 ± 0.15 kg body weight [BW]) were transported (loading + transport + unloading) for 11 h 40 min. Following transport, piglets were blocked into pens (n = 4 pigs/pen) by in utero and dietary treatments: in utero thermoneutral (IUTN) + control (C) diet (n = 10 pens), IUTN + ND (n = 10 pens), IUHS + C (n = 10 pens), and IUHS + ND (n = 10 pens). Treatment diets were fed from day 1 to 14 postweaning and transport (period 1), and the C diet was fed to all pigs from day 14 to 35 postweaning and transport (period 2). Production measures were taken in 7 d intervals to calculate average daily gain (ADG), average daily feed intake (ADFI), average daily net energy intake (ADEI), gain:feed, and gain:net energy intake. Blood samples were collected prior to transport, following transport, and on days 2, 7, 14, 28, and 35 postweaning and transport to analyze cortisol, glucose, insulin, and nonesterified fatty acids. Behavior was assessed through video-recording on days 3, 5, 8, 11, and 13 postweaning and transport. In period 1, ADG was reduced (P = 0.04; 20.0 g/d) in IUHS vs. IUTN pigs. Pigs fed ND diets had reduced ADFI (P = 0.02; 9.3%) compared with C diet-fed pigs during period 1, which resulted in similar ADEI (P = 0.23; 1,115 ± 35 kcal/d). During transport, cortisol was decreased (P = 0.03; 25.8%) in IUHS vs. IUTN pigs. On day 2, glucose was decreased (P = 0.01; 13.8%) in IUHS vs. IUTN pigs. No in utero treatment-related behavior differences were observed but lying was reduced (P = 0.03; 6.5%) and standing was increased (P = 0.04; 14.1%) in ND vs. C pigs overall. In summary, IUHS reduced growth performance in pigs following weaning and transport, and providing an ND diet did not rescue the lost performance.

Evaluating the Effects of In Utero Heat Stress on Piglet Physiology and Behavior Following Weaning and Transport.

The study objective was to determine whether in utero heat stress (IUHS) affects piglet physiology and behavior following common production practices. A total of 12 gilts were confirmed pregnant and allocated to either heat stress (HS; n = 6) or thermoneutral (TN; n = 6) conditions on day 30⁻60 of gestation. At weaning (22.5 ± 2.3 days of age), 1 boar and 1 barrow of median weight were selected from each litter and transported for approximately 7 h. Piglets were then blocked into pens (n = 2/pen) by in utero treatment (IUHS (n = 12) or in utero thermoneutral (IUTN, n = 12)) and sexual status (boar (n = 6/in utero treatment) or barrow (n = 6/in utero treatment)). Plasma cortisol, non-esterified fatty acids (NEFA), insulin and glucose were evaluated 1 day prior to transport (pre-transport) and immediately after transport (post-transport). Behavioral data were collected on day 1⁻7 for 60 min at four different time points each day. In utero heat stressed piglets exhibited reduced cortisol concentrations compared to IUTN piglets immediately post-transport (p = 0.04). Glucose concentrations were not affected by in utero treatment. Insulin concentrations were reduced in IUTN piglets post-transport compared to pre-transport (p = 0.002), but no differences were detected for IUHS pigs. Non-esterified fatty acids tended to be reduced overall for IUHS vs. IUTN pigs (p = 0.08). Overall, IUHS piglets performed more drinking behaviors (p = 0.02) and tended to perform more aggressive behaviors (p = 0.07) than IUTN piglets in the 7 days post-transport. In summary, there was some evidence for altered physiological and behavioral responses among IUHS piglets compared to IUTN piglets following weaning and transport.