Progress 07/01/22 to 06/30/23
Outputs
Target Audience:The target audiences that were reached during this reporting period were: producers, veterinarians, academics (most importantly in the fields of swine behavior & welfare, and pain assessment & management), and industry professionals within the U.S. Food and Drug Administration (FDA). By strategically selectingconferences and providing talks that target a broad audience, while also leaning on existing networks and active collaborations, we were able to disseminate our research results from this grant more widelywithin the United States. Looking ahead to 2024, we plan to target international conferences, including the annual congress of the International Society of Applied Ethology (ISAE) in Curitiba, Brazil and the International conference on the Welfare of Animals at Farm Level (WAFL) in Florence, Italy, to target audiences in Europe and South America. We feel strongly that to impact the most change in animal welfare, we must not only discuss our research results withindividuals here in the U.S., but also reach individuals outside of national borders. Changes/Problems:Personnel changes Dr. ZhoumengLin left Kansas State University. His role was to assist in characterizing the edible tissue residue depletion kinetics of firocoxib and assess potential toxicity in sows (Specific Aim 3). After much discussion with our pathology department and lab technicians, we have found this work can be done in-house and we no longer need to keep a salary line open for Dr. Lin. He has been removed from the grant. Dr. Michael Kleinhenz left Kansas State University. His role was to assist with blood collection in piglets. Dr. Eduarda Bortoluzzi has since joined Kansas State University. She has extensive experience with blood collection, behavior analysis, and animal welfare research in swine and cattle. She has agreed to replace Dr. Kleinhenz on this project. This personnel change has been approved Extension request Due to animal-phases occurring on two other USDA NIFA projects this summer (2020-67015-31540 and 2022-67015-36313), we were unable to complete the sow phase of this project, setting us back one-year. As such, I would like to request a no-cost extension from July 1, 2024- June 30, 2026. The reason for the two-year request is to provide enough time to complete the animal-phase and data analysis forObjectives 1 & 2(July 2024- June 2025) and to both complete the animal-phase and tissue residue analysis forObjective 3and give us the opportunity to disseminate the research results at conferences/meetings (July 2025- June 2026). This timeline will also ensure we have funds available to cover publication fees when the manuscripts are published. What opportunities for training and professional development has the project provided?Mentoring One full-time PhD student (Maria Lou): August 2020- present Tuition/fees/salary did not need to be paid until August 2021, which this grant has fully supported One dual DVM-MS student (Madeline Hall): January 2022- present One full-time veterinary scholar (Skylar Lierse): July-August2022 Not financially supported by this grant but involved in data collection/analysis One full-time research assistant (Jamie Gray): July-August 2022 One full-time research assistant (Tessa Van Buren): May-June 2023 One full-time veterinary scholar (Jessica Carnal): May-June 2023 Not financially supported by this grant but involved in data analysis Conferences This grant supported the lead PhD student, one research assistant, and one veterinary scholar to attend an Animal Care & Handling conference in Kansas City, MO from May 25-26, 2023 Memberships This grant has covered the PI and lead PhD student's annual fees to retain membership in the International Society of Applied Ethology (ISAE) How have the results been disseminated to communities of interest?Local Meetings Phi Zeta Research Day: March 7, 2023 (academics, veterinarians, graduate & veterinary students) Lead PhD student provided an oral presentation:Evaluation of oral firocoxib, administered to the sow and delivered transmammary to piglets, to alleviate pain associated with tail docking and castration Awarded 1st place in the Large Animal Clinical/Applied Research division Veterinary scholar provided a poster presentation:Oral administration of firocoxib in swine and evaluation of pain using infrared thermography Anatomy & Physiology Seminar Series:November 1, 2022(academics, veterinarians, graduate & veterinary students) Lead PhD student provided a seminar:Evaluation of oral firocoxib, administered to the sow and delivered transmammary to piglets, to alleviate pain after surgical castration and tail docking National Meetings Biomedical Sciences Seminar Series at Iowa State University: March 30, 2023(academics, veterinarians, graduate & veterinary students) PI travelled to ISU and provided a seminar, where this project and its application to industry were heavily featured:Novel pain assessment tools and mitigation strategies to improve the welfare of commercially-raised pigs Informally, this project and the resultsthus far have been discussed with colleagues and collaborators. For example, wehave discussed them with collaborators within the Food and Drug Administration (FDA), specifically involved in new animal drug evaluation for food-producing animals (ensuring compliance with FDA standards for data collection), colleagues within the American Association of Swine Veterinarians and the National Pork Board, and colleagues within a contract research facility who have since used our methods of oral firocoxib administration to sows. Recently, the PI discussed the medicated Gatorade method to an animal welfare colleague at the National Bio and Agro-Defense Facility (NBAF), who wanted to avoid repeatedly snaring sows to administer analgesic drugs. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we are planning to complete the remaining analysis of the data collected in the study described above, write the manuscript, and submit it for publication (priority; primary goal). We are also planning to launch our second study,Evaluating the efficacy of oral firocoxib to alleviate pain associated with parturition in the sow(primary goal). Our aim is to have the animal-phaseofObjective 1completed.Objective 2will largely focus on comparing previously validated methods of facial grimace collection in piglets with the methods employed in the study described above. We anticipate this will begin in the next reporting period, once the data analysis has been completed and the manuscript has been submitted for publication (secondary goal). It is unlikely thatObjective 3will be completed in the next reporting period. Research assistant positions will be made available earlynext year with the goal of hiring 2-3 students from May-August 2024, to assist with data collection and analysis. We are also looking at bringing on another dual DVM-MS student, who will primarily be focused on the sow phase of this project (Specific Aim 2; second study) andObjective 3.
Impacts
What was accomplished under these goals?
Objective 1 We completed the animal-phase of a study exploring which dose of transmammary-delivered firocoxib (3.0, 4.0, 5.0, or 6.0 mg/kg oral administration to the sow) was most effective at alleviating surgical castration and tail docking pain in piglets. Data was collected at the Kansas State University Swine Teaching and Research Center. Forty (40) sows nursing at least ten (10) viable piglets (5 male and 5 female;5±1 day old; minimum body weight = 1.0 kg) were enrolled. Sows were randomly assigned to 1 of 5 treatment groups (n=8 sows/treatment): (1) 3.0 mg/kg oral firocoxib; (2) 4.0mg/kg oral firocoxib; (3) 5.0mg/kg oral firocoxib; (4) 6.0mg/kg oral firocoxib; (5) placebo. Twenty-four (24) hours prior to study start, baseline measurements were taken of each piglet, including body weight, blood (for cortisol and firocoxib concentration), infrared thermography, behavior, facial grimacing, and gait score (specifics of each of these measures are outlined below). On Day 0, sows received their assigned dose of oral firocoxib. Equioxx tablets were crushed using a pill crusher until they were a fine powder and then dissolved in Gatorade (flavour: Fierce Grape). The medicated Gatorade was then placed in a syringe with a drench tip and provided orally to the sow. At the 12h post-administration time point, piglets within a litter were removed and placed in a holding cart. They received a number on their back using a black permanent marker for identification throughout the study. Male piglets (n=4/litter; n=160 total for the study) were surgically castrated using a scalpel and tail docked using side pliers. Female piglets(n=4/litter; n=160 total for the study) were only tail docked until side pliers. One male and one female piglet (n=2/litter; n=80 total for this study) served as control animals (sham) and did not undergo any painful procedure. Vocalizations A camera with a microphone was set-up on a tripod to record procedure-associated vocalizations of piglets. The resulting audio files were analyzed using Raven Pro v 1.6 software, with the vocal parameters of interest beingduration (sec),energy (dB),peak frequency (Hz), andpeak amplitude (µ). There was no difference in vocalizations between treatment groups, suggesting none of the treatment groups were able to significantly reduce painat the time of surgical castration and tail docking. We did find that surgical castration elicited vocalizations of higher energy and peak amplitude (i.e., "louder" vocalizations) compared to tail docking. Similarly, we found that male pigletsproduced calls of longer duration, higher energy and peak amplitude (i.e., "longer and louder" vocalizations) than female piglets. Behavior A camera set-up on a tripod was used to record piglet behavior post-processing while they were in the farrowing pens (prior to handling piglets for other outcome measure assessment). Behavior was collected at 0, 1, 2, 4, 7, 24, 36, and 48h post-processing. Videos are currently being scored by two experienced observers using BORIS software and a detailed ethogram. Methodology used: focal-animal, continuous sampling for 15 min at each time point. Infrared thermography (IRT) IRT images of the piglets'scrotum and tail tip were collected at the following post-processing time points: 1, 7, 24, 36, and 48h using an infrared camera (FLUKE TiX580). This served as a non-invasive measure of inflammation at the surgical site. Piglets were held gently against the body to facilitate data collection and minimize stress. The IRT images were analyzed using SmartView v 4.3 software. At 24h post-processing, castrated piglets from the 3.0 mg/kg firocoxib group had significantlylower scrotal temperatures than piglets from the 5.0 and 6.0 mg/kg firocoxib groups, which suggests there was less inflammation at the surgical site. Plasma firocoxib and cortisol concentrations At 1 and 24h post-processing, 4.0 mL whole blood was collected from a subset of piglets (n=3/litter; n=120 total for this study) via jugular venipuncture using a 21G, 1-1.5" needle. Laboratory technicians at Kansas State University have processed the samples (using ultra-high pressure liquid chromatography coupled with mass spectroscopy; UPLC-MS) andcompleted the plasma firocoxib assays(cortisol assays are ongoing). The mean firocoxib concentrations at the level of the piglet were as follows: (1) 3.0 mg/kg firocoxib group = 119.3±24.9 ng/mL; (2) 4.0 mg/kg firocoxib group = 200.1±30 ng/mL; (3) 5.0 mg/kg firocoxib group = 219.5±22 ng/mL; (4) 6.0 mg/kg firocoxib group = 226.7±30.6 ng/mL. Piglets in the 5.0 and 6.0 mg/kg firocoxib groups had significantly higher firocoxib concentrations than piglets in the 3.0 mg/kg firocoxib group; however, there were no significant differences in concentration among piglets in the 4.0, 5.0, or 6.0 mg/kg firocoxib groups. This suggests that there may be a threshold in how much firocoxib is able to cross the blood-milk barrier and increasing firocoxib doses to the sow greater than 4.0 mg/kg will not result in significantlymore drug at the level of the piglet (i.e., there is a point where it will not increase in a dose-dependent manner). Firocoxib concentrations across all treatment groups remained stable at 1and 24h post-processing (13 and 36h post-dosing sows), demonstrating the long-acting ability of firocoxib as an NSAID and suggests that dosing sows 12h pre-processing piglets provided sufficient time for adequate drug concentration to reach the level of the piglet. Gait analysis Piglets were walked across a pressure mat (Tekscan) at 1, 7, 24, 36, and 48h post-processing. Strideway software is being used to assess the following gait parameters:stance time (sec),stride length (cm),peak vertical force (kg),vertical impulse (kg*sec),peak pressure (kg/cm2),gait distance (cm), andgait velocity (cm/sec). Data analysis is ~50% complete; there are no preliminary results to share at this time. Facial grimace A HD webcam was set-up at the end of the pressure mat to capture piglet facial expressions as they walked across. Therefore, the time points aligned with those described in the "gait analysis" section.This is a novel way to collect facial grimace data and will be compared to previously developedmethods for validity inObjective 2. Still-images of the piglet's face are being pulled from the collected video using VLC Media Player and will be scored by five individuals using the Piglet Grimace Scale (Viscardi et al., 2017). Interobserver reliability will be assessedbetween all scorers prior to statistical analysis. Body weight At 21 days of age (i.e., at weaning), piglets were weighed and their average daily gain was calculated. Piglets in the 3.0 mg/kg firocoxib group gained significantly more weight than the piglets from the 4.0, 5.0, and 6.0 mg/kg firocoxib groups. At this point in the analysis, it appears that 3.0 mg/kg firocoxib, administeredorally to the sow and delivered transmammary to her nursing piglets, may be effective in increasing piglet weaning weights and reducing inflammation in surgically castrated piglets. The benefits of transmammary-delivered firocoxib to the welfare of piglets undergoing surgical castration and tail docking will be verified after the full analysis of all study outcomes are completed.
Publications
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:The target audiences that were reached during this reporting period were: livestock producers, veterinarians, research scientists, and animal health officials within the U.S. As the data collection phase for this project had not been initiated at this time, the conversations with these individual groups were largely focused on the concept and benefits oftransmammary drug delivery in production swine. We also shared preliminary data from previous work as a "proof of concept" rationale for embarking on this larger inquiry into swine pain management. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Mentoring One full-time PhD student (Maria Lou): August 2020- present Tuition/fees/salary did not need to be paid until August 2021, which this grant has fully supported One dual DVM-MS student (Madeline Hall): January 2022- present One full-time veterinary scholar (Skylar Lierse): May-June 2022 Not financially supported by this grant but involved in data collection/analysis One full-time research assistant (Jamie Gray): May-June 2022 Conferences This grant supported the PI and lead PhD student to virtually attend the Congress of the International Society of Applied Ethology (ISAE) from August 2-6, 2021 This grant supported the PI and lead PhD student to virtually attend the International Conference on the Assessment of Animal Welfare at the Farm and Group Level (WAFL) from August 16-19, 2021 This grant supported the PI and lead PhD student to attend the North American Regional Meeting of the International Society of Applied Ethology (NA-ISAE) in Davis, CA from April 29-30, 2022 Memberships This grant has covered the PI and lead PhD student's annual fees to retain membership in the International Society of Applied Ethology (ISAE) How have the results been disseminated to communities of interest? National Meetings North American Regional Meeting of the International Society of Applied Ethology (NA-ISAE): April 29-30, 2022 (academics, industry professionals, veterinarians, producers, graduate students) PI travelled to Davis, CA and provided an oral presentation:Evaluation of firocoxib, delivered transmammary to piglets, to alleviate pain associated with elective husbandry procedures United States Animal Health Association (USAHA): October 8, 2021 (academics, industry professionals, veterinarians, producers) PI provided an oral presentation virtually, where the novelty of transmammary-drug delivery was discussed:Pain management in livestock species What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we are planning to complete the animal (piglet) phase of the project, that will partially satisfyObjectives 1and2. We also plan to complete most of the data analysis for the outcome measures collected. We hope you have a preliminary idea of which dose of transmammary-delivered firocoxib (3.0, 4.0, 5.0, or 6.0mg/kg oral administration to the sow) was most effective at alleviating surgical castration and tail docking pain.
Impacts
What was accomplished under these goals?
Objective 1 & 2 The IACUC protocol (#4762) was submitted and approved. The animal (piglet) phase of this study will begin in July 2022. In preparation, personnel were hired and trained on low-stress swine handling and how to collect the planned outcome measures. We put together drug protocols, standard operating procedures (SOPs), and data collection forms for: body weight, blood collection, infrared thermography, behavior, facial grimacing, and pressure mat. Equipment was tested to ensure everything is in good working order.
Publications
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