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Page 1 / 20 Topical Wound-care Products and Their Effects on Healing, In ammatory Biomarkers, and Growth in Piglets Undergoing Castration Laya Kannan Silva Alves Universidade de São Paulo Monique Danielle Pairis-Garcia North Carolina State University Juliana Bonin Ferreira North Carolina State University Victoria Rocha Merenda North Carolina State University Rubia Mitalli Tomacheski Washington State University Pedro Henrique Esteves Trindade Michigan State University Christopher Siepker Iowa State University Magdiel Lopez-Soriano University of Missouri Research Article Keywords: acute phase proteins, animal welfare, piglet mortality, thermography, weaning, wound healing Posted Date: October 12th, 2025 DOI: https://doi.org/10.21203/rs.3.rs-7722871/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported.

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Page 2 / 20 Abstract Surgical castration is a routine management procedure in swine production that raises welfare concerns due to pain, in ammation, and risk of post-procedure complications. Topical products are commonly applied to castration wounds, but their e cacy in promoting healing and reducing in ammation has not been systematically evaluated. This study investigated the e cacy of ve commercially available topical protective products on wound healing, in ammatory responses, and growth performance in piglets undergoing surgical castration. One hundred and ninety piglets were assigned to one of six groups: Iodine, Oinkment®, PhytoCare®, Vetericyn®, Zinc Oxide, or intact controls (NoCast). Treatments were applied immediately after castration (D1). Body weights were recorded at baseline (D0) and at weaning. Blood samples were collected on days 0 (baseline), 7, and 14 for analysis of prostaglandin E ₂ (PGE ₂ ) and haptoglobin. Infrared thermography (IRT) was used to assess scrotal surface temperature. Histological evaluation of wound healing was performed on subsets of piglets on days 7 and 14. No treatment effects were observed on body weight or pre-weaning survival; castrated piglets grew similarly to intact controls. Concentrations of PGE ₂ declined over time ( P < 0.001) but did not differ between treatments, suggesting it may have limited utility as an in ammatory biomarker in neonatal pigs. Haptoglobin concentrations increased across all groups by days 7 and 14, including intact controls, indicating limited speci city for castration-related in ammation. In contrast, IRT consistently distinguished castrated from intact piglets, supporting its potential as a non-invasive indicator of in ammatory responses. Histological evaluations showed expected

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castrated from intact piglets, supporting its potential as a non-invasive indicator of in ammatory responses. Histological evaluations showed expected time-dependent healing progression, with epidermal thickness correlating with wound severity, but no treatment effects were found. None of the tested topical products improved wound healing or reduced systemic in ammation under a single-application protocol. While safe and without adverse effects on growth, their bene ts appear limited under the study conditions. Future research should explore repeated applications, microbial wound presence, and behavioral indicators to better evaluate post-castration wound-care strategies. INTRODUCTION Castration is a common procedure performed on swine farms in the United States to prevent unwanted breeding, reduce aggression and improve meat quality (Weiler et al., 2021 ; Breitenlechner et al., 2024 ). This procedure is painful and has been identi ed as a signi cant welfare concern both from within the US swine industry as well from the global public’s perspective (Morgan et al., 2019 ; Miller et al., 2023 ). Surgical castration is typically conducted within the rst 7 days of the piglet’s life, utilizing an open technique where two incisions are made in the scrotal skin to expose and remove the testicles (Hokkanen et al., 2025 ). After the testicles are removed, the spermatic cords are severed, and the incisions are left open to heal naturally without sutures (Rault et al., 2011 ; Coutant et al., 2022 ). Surgical castration induces marked physiological changes in piglets, including acute stress response such as increased plasma cortisol concentrations (Nixon et al., 2021 ), and tissue-level in ammation indicated by acute phase protein secretion (Charlie-Silva et al., 2019). In

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ortisol concentrations (Nixon et al., 2021 ), and tissue-level in ammation indicated by acute phase protein secretion (Charlie-Silva et al., 2019). In addition, the open healing process increases the risk of morbidity and mortality risk during the pre-weaning phase (Guay et al.,

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Page 3 / 20 2013 ; Morales et al., 2017 ; Schmid et al., 2021 ), with potential complications including excessive bleeding, infection, delayed wound healing, and herniation at the incision site (AVMA, 2013; Viscardi et al., 2020 ; Schmid et al., 2021 ). These risks are particularly elevated in lighter pigs or those with comorbidities (Telles et al., 2016 ; Morales et al., 2017 ). In addition to the physiological complications and increased risk of morbidity, these outcomes contribute to an overall negative welfare state for the piglet. According to the Five Domains model (Mellor & Beausoleil, 2015 ), such impacts can be systematically interpreted across multiple dimensions of welfare, including health, environment, and nutrition, with each in uencing the piglet’s affective state. This framework provides a structure method to evaluate how physical and functional disruptions, such as tissue injury and impaired growth, ultimately shape the mental experiences of the animal. In the context of castration, the procedure directly compromises the health domain (through pain, in ammation, and infection risk), the environment domain (via exposure of open wounds to pathogens), and the nutrition and performance domain (through potential reductions in growth due to morbidity). Despite the welfare and economic implications of castration, limited research has been conducted evaluating effective and practical strategies for improving post-castration wound healing and decreasing pre-weaning mortality and morbidity. To date, the most common post-castration protocol utilized on- farm is the use of a topical iodine spray applied to the incision site, primarily serving as a disinfectant to reduce bacterial load and minimize environmental contamination (Gooch, 2010 ; Jacobs & Neary, 2020 ).

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ion site, primarily serving as a disinfectant to reduce bacterial load and minimize environmental contamination (Gooch, 2010 ; Jacobs & Neary, 2020 ). However, no studies to date have evaluated this method nor compared it to alternative wound care products and their effects on the physiological response of the piglet during the wound healing process. To reduce piglet loss associated with castration-related death, identifying an effective product that improves wound healing rates and decreases infection associated with castration is critical. Therefore, the objective of this study was to evaluate the e cacy of ve topical protective products on wound healing, physiological response, in ammation, and performance in castrated piglets. MATERIAL AND METHODS Housing and animals This study was approved by the Institutional Animal Care and Use Committee of North Carolina State University (IACUC protocol 20–113). The experiment was conducted on a commercial sow farm in the southeastern United States during the summer. Sows and piglets were housed in individual farrowing crates within tunnel-ventilated, fully slatted farrowing rooms maintained at an average temperature of 22º ± 1.0 ºC. Temperature and ventilation were controlled using a computerized system. Each farrowing crate measured 2.5 m × 0.7 m, with an additional piglet area (2.5 m × 1.3 m). Heat mats were provided for piglets and maintained at approximately 30–35°C. Lighting was provided from 0600 to 1700 h. Feed and water were offered ad libitum to both sows and piglets.

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Page 4 / 20 Animal care and handling followed the Guide for the Care and Use of Agricultural Animals in Research and Teaching (FASS, 2020). Surgical castration was a routine farm practice; therefore, male piglets enrolled in this study were not castrated solely for research purposes. Experimental design and treatment administration A total of 190 Large White x Duroc male piglets from 51 litters were enrolled in this study (study duration = 21 days; Fig. 1 ). At enrollment (D0), piglets were individually identi ed using ear tags (All ex Global Piglet ear tags, All ex Livestock Intelligence, Madison, WI), weighed, and randomly assigned to one of six treatment groups: I (Iodine; CSR Company, La Vist, NE): male piglets (n = 32) surgically castrated with topical application of iodine at the incision site immediately post-procedure. O (Oinkment ® ; Animal Science Products; Nacogdoches, TX, USA): Male piglets (n = 32) surgically castrated with topical application of Oinkment® at the incision site post-procedure. ZO (Zinc oxide Ointment USP ® ; Rugby Laboratories, Livonia, MI, USA): Male piglets (n = 32) surgically castrated with topical application of Zinc oxide Ointment USP® at the incision site post- procedure. PC (PhytoCare Swine Skin Recovery & Care ® ; Precision Health Technologies, Brookings, SD, USA): Male piglets (n = 31) surgically castrated with topical application of PhytoCare Swine Skin Recovery & Care® at the incision site post-procedure. VP (Vetericyn Plus ® ; Vetericyn, Rialto, CA, USA): Male piglets (n = 31) surgically castrated with topical application of Vetericyn Plus® at the incision site post-procedure. NoCast (Non-castrated): Intact male piglets (n = 32) subjected to sham castration with topical application of iodine to the testicles post-sham

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edure. NoCast (Non-castrated): Intact male piglets (n = 32) subjected to sham castration with topical application of iodine to the testicles post-sham procedure. All treatments were applied immediately post-castration (D1). Liquid treatments (iodine, Phytocare, Vetericyn) were administered using a spray bottle (1–3 ml per piglet), while paste products (Zinc Oxide Ointment and Oinkment) were applied via a gloved hand (2–3 grams per piglet). Castration procedure Castration was performed on D1 by a trained researcher (MLS) with over 10 years of swine production experience. Piglets were restrained by holding both hind legs with the head down. A sterile scalpel blade was used to make two vertical incisions through the scrotal skin over each testicle. The testicles were exteriorized, the spermatic cords severed, and the testicles removed by traction. Piglets in non-castrated group underwent a sham procedure in which they were restrained similarly, but only external pressure

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Page 5 / 20 was applied to the scrotal area without making an incision. The same researcher performed both castration and sham procedures to ensure consistency. Data collection Piglet performance Piglet body weight was recorded at enrollment (D0), on castration day (D1), and subsequently on days 7, 14, and 21 (weaning). Average body weight (BW) and average daily gain (ADG) were calculated for each treatment at all time points based on live pig weight. Pre-weaning mortality was also assessed throughout the 21-day nursing period. Physiological response Blood samples were collected to assess prostaglandin E 2 (PGE 2) and haptoglobin concentration for each piglet (n = 190) 24h prior to castration (D0 - baseline), 7-day post-castration, and 14 days post-castration (Fig. 1 ). Blood collection was performed using a technique previously described by Dove and Alworth ( 2015 ) and Lopez-Soriano et al., ( 2022 ). Samples were obtained via orbital sinus puncture using an 20G Excel® disposable hypodermic needle (Exel International, Quebec, Canada) and collected into 4ml BD® red-top vacutainer serum tubes (Med Vet International, Mettawa, IL). The tubes were immediately placed in a cooler and centrifuged (2,000 × g for 15 min at 4°C) within eight hours of collection to separate serum. The serum was then aliquoted into 1.5ml Axygen® microcentrifuge tubes (Axygen Scienti c, Corning, NY) at − 80°C. The assays for the biomarker analysis were completed six to nine months post-collection. PGE 2 concentrations were measured using a commercial enzyme-linked immunosorbent assay (ELISA; catalog No. 514531; Cayman Chemical) following the method described by Giorgi et al., ( 2011 ). Brie y, serum samples were puri ed by adding ice-cold acetone (4x the serum volume), followed by incubation

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thod described by Giorgi et al., ( 2011 ). Brie y, serum samples were puri ed by adding ice-cold acetone (4x the serum volume), followed by incubation at − 20° C for 30 minutes and centrifugation (3,000 x g for 5 minutes). The supernatant was transferred to a 13 x 100-mm glass tube, evaporated using a CentriVap concentrator (Labconco), and reconstituted to the original serum volume with kit buffer. An aliquot of the reconstituted sample was derivatized with adjusted kit components and the manufacturer’s protocol were followed. Samples were analyzed in duplicate, and absorbance was measured at 405 nm following 60 minutes of development (SpectraMax i3; Molecular Devices). The mean PGE 2 concentration of a reference sample used for repeatability assessment was 12.42 pg/ml (range: 9.90–15.60 pg/mL), with an inter-assay coe cient of variation of 8.61% (Merenda et al., 2022 ). Haptoglobin concentrations were determined using the Tridelta Phase® Haptoglobin Assay (catalog No. TP-801; Tridelta Development Ltd., Maynooth, Ireland). The assay utilized Haemoglobin (reagent 1), Chromogen (reagent 2), calibrator/sample diluent, and calibrator (microplate method). A standard curve was generated for each assay using haptoglobin standards at 2.5, 1.25, 0.625, 0.312, and 0 mg/mL. The

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Page 6 / 20 assay procedure involved dispensing reagents 1 and 2 into storage vessels within the instrument, followed by aliquoting samples, controls, and calibrators into the appropriate sample cuts. Absorbance was then measured at 600–630 nm while the calibration curve developed. Results were interpreted by calculating the mean absorbance for each sample, control, or standard. A calibration curve was generated by plotting absorbance (600–630 nm) against haptoglobin concentration (mg/mL), and a smooth curve was tted through the data points. For porcine samples, expected haptoglobin values were categorized as follows: normal range (0.00-2.2- mg/mL) and acute phase range (3.00–8.00 mg/mL). The intra assay and inter assay coe cients of variation were 5.8% and 4.9%, respectively. In ammatory response Infrared thermography (IRT) was used to detect changes in skin temperature associated with in ammation. Image collection followed the methodology described by Bates et al., ( 2014 ), utilizing a portable infrared camera (Degree2Act). Temperature changes were analyzed by comparing the piglets' skin temperature at different time points: 24 hours before castration (D0 - baseline), immediately after castration (D1), and at 7- and 14-day post-castration. Histopathological response To assess histopathological changes at the incision site, punch biopsies were collected on days 7 and 14 of the trial from ve pigs per treatment group. The randomly selected piglets received pain control consisting of 1 mL of 2% lidocaine applied in the inguinal area and unixin meglumine (2.2 mg/kg) administered intranasally 20 minutes prior to sample collection. Punch biopsies were obtained using a 5.0 mm round-tipped cutting tool (Scienti c Labwares Disposable Punch Biopsy®, Gainesville, VA,

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ample collection. Punch biopsies were obtained using a 5.0 mm round-tipped cutting tool (Scienti c Labwares Disposable Punch Biopsy®, Gainesville, VA, USA) from the scrotal skin at the castration incision site. The samples were collected by applying slight downward pressure and rotating the device clockwise. Once the skin was punctured, the punch device was carefully removed, and the skin samples were grasped with forceps. The underlying fat was then separated using scissors. Samples were immediately xed in 10% formalin (Hackworth, 2019 ). Histological samples were processed using Masson’s trichrome staining, and a board-certi ed pathologist evaluated them based on the variables described in Table 1 .

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Page 7 / 20 Table 1 Wound healing objective criteria (adapted from Santos et al., 2021 and van de Vyver et al., 2021 ). Score: 0 1 2 3 Epidermal ulceration intact (< 5%) partial (5– 25%) ulceration (25– 50%) complete ulceration (> 50%) Epidermal thickness index 1 normal (95– 105%) mild (< 110%) moderate (110– 120%) marked (> 120%) Serocellular crusting absent mild (25– 50%) moderate (50– 75%) marked (> 75%) In ammatory in ltrate minimal mild moderate marked Granulation tissue none/minimal mild (10– 25%) moderate (25– 50%) proliferative (> 50%) Dermal hemorrhage absent mild moderate marked 1 ETI = (average thickness of epidermis in wound area]/[average thickness of epidermis in uninjured skin) × 100 Statistical analysis All analyses were conducted using RStudio (version 2024.04.0 Build 735; R Core Team, 2024 ). The experimental unit for performance, physiological, and in ammatory outcomes was the individual piglet, while histopathological data were analyzed at the biopsy level (n = 5 piglets per treatment per timepoint). PGE 2 and haptoglobin concentrations were analyzed using generalized linear mixed models with a Gamma distribution and log link. Fixed effects included treatment, timepoint (day), and their interaction. Random intercepts accounted for piglets nested within litters. Baseline concentrations of the biomarkers were included as covariates, along with piglet weight at enrollment, baseline infrared body surface temperature (IRTd0), sow parity, and litter characteristics (born alive, stillbirths, and mummi ed piglets). Baseline haptoglobin levels were a strong positive predictor of post-treatment concentrations ( P < 0.001), supporting their inclusion in the model as a covariate. Infrared thermography and piglet body weight data were analyzed

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t concentrations ( P < 0.001), supporting their inclusion in the model as a covariate. Infrared thermography and piglet body weight data were analyzed using linear mixed-effects models with the same xed and random effect’s structure. Litter was modeled as a random intercept. Pre-weaning mortality was compared across treatments using Chi-square or Fisher’s exact tests, as appropriate. Histopathological scores were analyzed using non-parametric Kruskal–Wallis tests followed by Dunn’s post hoc comparisons. A linear regression model was also tted to the total histological score to explore associations with treatment, day, treatment x day interaction, and covariates. Least-squares means

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Page 8 / 20 (LSMeans) were estimated, and pairwise comparisons were adjusted for multiple testing using Holm’s method. Statistical signi cance was set at α = 0.05. RESULTS Piglet Performance Sow reproductive parameters or piglet pre-weaning mortality ( P > 0.05) did not differ between treatments; thus, these variables were excluded from subsequent models. Weight relative to castration is depicted in Fig. 2. Treatment and the interaction between treatment and day did not affect body weight over time ( P > 0.05). However, a signi cant effect of day was observed ( P < 0.05), indicating consistent growth across the 21-day period. Physiological response Plasma prostaglandin E ₂ (PGE ₂ ) Plasma PGE ₂ metabolite concentrations are presented in Fig. 3 (see Supplementary Table S1 for detailed LSM ± SEM values). A signi cant effect of timepoint was observed, with PGE ₂ concentrations decreasing on day 7 ( P < 0.001) and day 14 ( P < 0.001) relative to baseline (day 0). No signi cant effects of treatment ( P > 0.05) or treatment × day interaction ( P > 0.05) were detected. Plasma haptoglobin concentrations Plasma haptoglobin concentrations (mg/dL) are shown in Fig. 4 (see Supplementary Table S2 for detailed LSM ± SEM values). No signi cant effects were detected for treatment ( P > 0.05) or for the treatment × timepoint interaction ( P > 0.05). However, a main effect of timepoint was observed, with haptoglobin levels elevated on both day 7 ( P < 0.001) and day 14 ( P < 0.001) compared to day 0. In ammatory response Infrared thermography (IRT) measurements are presented in Fig. 5. A signi cant effect of treatment ( P < 0.05), day ( P < 0.05), and the treatment × day interaction ( P < 0.05) was observed. Overall, NoCast piglets exhibited consistently lower skin

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( P < 0.05), day ( P < 0.05), and the treatment × day interaction ( P < 0.05) was observed. Overall, NoCast piglets exhibited consistently lower skin surface temperatures compared to all castrated groups, regardless of the topical treatment applied, across all evaluated timepoints. Histopathological response Histopathological measurements collected on days 7 and 14 are summarized in Table 4 . No signi cant effects of treatment ( P > 0.05) or treatment × day interaction ( P > 0.05) were detected for any of the evaluated histological parameters. However, a signi cant effect of timepoint was observed ( P < 0.05) for epidermal thickness index, ulceration, serocellular crusting, granulation tissue, and total combined histological score, with all measures showing a reduction on day 14 compared to day 7, consistent with normal wound healing progression.

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Page 9 / 20 DISCUSSION This study investigated the e cacy of ve commercially available topical products in mitigating in ammation and promoting wound healing following surgical castration in piglets under commercial conditions. To our knowledge, no prior research has evaluated the effect of topical formulations on the wound healing process in castrated piglets. Similar work to this study has largely focused on topical formulations used to reduce acute pain at the time of castration (Sutherland et al., 2010 ; Gottardo et al., 2016 ; Lomax et al., 2017 ; Sheil et al., 2020 ) without a direct evaluation of how such products did or did not effectively support tissue repair or healing. Therefore, this study can be used as foundational knowledge for exploring wound healing and repair of commercially available topical products for swine processing procedures like castration. Across treatments, no effects were observed on growth performance or pre-weaning survival, suggesting that topical applications did not negatively impact pre-weaning performance. However, castrated piglets maintained similar weights to intact piglets that did not undergo castration. Although it is well established that castration causes growth setbacks (Telles et al., 2016 ; Morales et al., 2017 ), our results did not capture these differences. Our ndings are similar to Kielly’s et al. (1999) who reported weight loss in the rst days post-castration but no differences at weaning. In our study, piglets were weighed on a weekly basis, which may have limited our ability to detect subtle transient changes in growth performance occurring within shorter intervals post-procedure. Future studies should include more frequent weight records (e.g., daily in the immediate post-castration period) to better capture

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als post-procedure. Future studies should include more frequent weight records (e.g., daily in the immediate post-castration period) to better capture growth trajectories. In addition to the absence of differences in performance measures, in ammatory measures also did not differ among treatment groups, and PGE ₂ levels declined across all groups, including non-castrated piglets. This nding aligns with previous studies (Nixon et al., 2021 ; Lopez-Soriano et al., 2022 ) and suggests that PGE ₂ is not a reliable post-operative in ammatory biomarker in neonatal pigs (Everaert et al., 2017 ; Merenda et al., 2024 ). Its elevated baseline levels may re ect physiological processes such as intestinal development, microbial colonization, or transient neonatal immune activity of pre-weaned piglets, despite undergoing castration (Merenda et al., 2024 ). These results indicate that while PGE ₂ plays a central role in in ammation, its diagnostic value in early-life castration models is limited. Similar to PGE 2 results, haptoglobin did not demonstrate sensitivity as an indicator of castration in ammation. Haptoglobin is an acute-phase protein synthesized in the liver and commonly used as an indicator of systemic in ammation and tissue injury (Merlot et al., 2013 ). In this study, haptoglobin concentrations increased signi cantly by Day 7 and remained elevated on day 14 across all groups, including intact piglets. This pattern indicates that the observed response may not be attributable to castration-related in ammation. Instead, increases could re ect other early-life factors, such as social stress, androgen-driven immune modulation in intact males (Fardisi et al., 2023 ), or subclinical infections (Saco & Bassols, 2022 ). These ndings highlight the multifactorial nature of

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ation in intact males (Fardisi et al., 2023 ), or subclinical infections (Saco & Bassols, 2022 ). These ndings highlight the multifactorial nature of haptoglobin responses in neonatal pigs and suggest it may lack speci city as a biomarker for castration associated in ammation.

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Page 10 / 20 Future work should consider alternative biomarkers with greater sensitivity such as C-reactive proteins, serum amyloid A (Pomorska-Mól et al., 2013 ) or pro-in ammatory cytokines such as IL-1 β and TNF- α (Llamas Moya et al., 2008 ), to more precisely characterize the in ammatory response to castration. Infrared thermography (IRT) proved to be a non-invasive and sensitive method for distinguishing in ammatory responses between castrated and intact piglets, however there was no treatment effect, demonstrating that the products did not drive changes to scrotal temperature, but the castration procedure itself. Castrated piglets consistently exhibited higher skin surface temperatures than their intact counterparts. This is likely due to local in ammation and increased blood perfusion in response to tissue injury (Korkmaz et al., 2017 ), consistent with previous work demonstrating that IRT can detect thermal changes associated with castration or other surgical wounds in livestock and companion animals (Stewart et al., 2010 ; Viscardi et al., 2020 ; Bergamasco et al., 2021 ; Saidu et al., 2023 ). Additional considerations should also include the role of testosterone on scrotal temperatures, with previous work demonstrating the role of testosterone in reducing skin perfusion, thus decreasing surface temperature (Farhat et al., 1995 ). Thus, future studies could consider unilateral castration to determine the role of an intact testicle on scrotal surface temperature. Histopathological evaluation revealed time-dependent improvements in wound healing between days 7 and 14, including reductions in ulceration, crusting, granulation tissue, and overall wound scores of all piglets. Epidermal thickness was the only histological variable correlated with total wound

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sting, granulation tissue, and overall wound scores of all piglets. Epidermal thickness was the only histological variable correlated with total wound severity, suggesting it may be a useful proxy for local in ammatory activity (Greaves, 2012 ) and could potentially serve as a standalone measure for wound healing. Although histopathologic results demonstrated wound healing improvement over the two weeks, no treatment effect was observed. These ndings indicate that none of the tested products signi cantly impaired or enhanced the healing process under the application conditions used. The lack of treatment effect is an important result for the global swine industry to consider. Iodine is the most widely used product for post-castration wound care in pig production systems, valued for its low cost, ease of application and presumed antimicrobial action (Jacobs & Neary, 2020 ; King-Podzaline et al., 2024 ). Despite its routine use, there is little to no previous evidence demonstrating its e cacy, and the results from this study indicate no measurable healing bene t compared to alternative products. Re- evaluating standard practices performed on farms is needed to ensure protocols are in line with scienti c evidence and management practices are being performed to truly bene t the health and welfare of the animal. Limitations of this work should be acknowledged. First, the lack of treatment effects may re ect the single application protocol and limited sample size of the study. A larger sample size and an experimental design permitting repeated or long-term application of the product should be considered. In addition, while physiological and histological measures were included, behavioral and microbiological assessments were not incorporated, limiting the scope of welfare

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physiological and histological measures were included, behavioral and microbiological assessments were not incorporated, limiting the scope of welfare and infection-related insights. Finally,

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Page 11 / 20 our study design did not include a group of castrated piglets, receiving no wound care, preventing direct evaluation of whether topical treatments confer advantages compared to no intervention. CONCLUSIONS None of the tested topical products signi cantly improved wound healing or reduced in ammation following surgical castration in piglets. Haptoglobin and prostaglandin E 2 were not effective biomarkers of castration in ammation, but infrared thermography effectively detected post-castration in ammation. Histological healing progressed over time regardless of treatment, with epidermal thickness serving as a key indicator of wound response. While the products were safe and did not impair growth, their bene ts under a single-application protocol were limited. Future research should increase sample size, repeated applications, behavioral indicators suggestive of wound healing, and microbial assessment of incisional sites to better evaluate product e cacy on wound healing. Declarations Acknowledgements The author LKSA acknowledges the São Paulo Research Foundation (FAPESP) for nancial support through a research fellowship (Grant nº 2021/08217-5; 2023/07961-8). Author contributions LKSA: data visualization, writing – original draft, writing - review and editing nal document. MDPG: conceptualization, methodology, data collection, writing – review and editing nal document, supervision, and funding acquisition. JBF: methodology, data visualization, writing – review and editing nal document. VRM: methodology, statistical analysis, data visualization, writing - review and editing nal document. RMT: methodology, data collection, writing - review and editing nal document; PHET: methodology, statistical analysis, data visualization, writing - review and

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dology, data collection, writing - review and editing nal document; PHET: methodology, statistical analysis, data visualization, writing - review and editing nal document; CS: methodology, data visualization, writing – review and editing nal document. MLS: conceptualization, methodology, on-farm data collection, statistical analysis, data visualization, writing original draft, writing – review and editing nal document. All authors have reviewed the manuscript. Funding This research was funded by Animal Science Products. Data availability statement

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Page 12 / 20 The data that support the ndings of this study are available from the corresponding author upon reasonable request. Competing interests’ statement The authors declare no competing interests. References 1. AVMA (American Veterinary Medical Association). (2013). Literature review on the welfare implications of swine castration. May 29, 2013. Available at: https://www.avma.org/KB/Resources/LiteratureReviews/Documents/swine_castration_bgnd.pdf. 2. Bates, J.L., Karriker, L.A., Stock, M.L., Pertzborn, K.M., Baldwin, L.G., Wulf, L.W., Lee, C.J., Wang, C., Coetzee, J.F. (2014). Impact of transmammary-delivered meloxicam on biomarkers of pain and distress in piglets after castration and tail docking. PLoS One 9:e113678. doi: 10.1371/journal.pone.0113678 3. Bergamasco, L., Edwards-Callaway, L.N., Bello, N.M., Mijares, S.H., Cull, C.A., Rugan, S., Mosher, R.A., Gehring, R., Coetzee, J.F. (2021). Unmitigated surgical castration in calves of different ages: Cortisol concentrations, heart rate variability, and infrared thermography ndings. Animals 11:2719. doi: 10.3390/ani11092719 4. Breitenlechner, A., Bünger, M., Ruczizka, U.K., Dolezal, M., Auer, U., Buzanich-Ladinig, A. (2024). Comparison between intramuscular and intranasal administration of sedative drugs used for piglet castration. Animals 14:2325. doi: 10.3390/ani14162325 5. Carroll, J.A., Berg, E.L., Strauch, T.A., Roberts, M.P., Kattesh, H.G. (2006). Hormonal pro les, behavioral responses, and short-term growth performance after castration of pigs at three, six, nine, or twelve days of age. J. Anim. Sci. 84:1271-1278. doi: 10.2527/2006.8451271x . Coutant, M., Malmkvist, J., Kaiser, M., Foldager, L., Herskin, M.S. (2022). Piglets’ acute responses to local anesthetic injection and surgical castration:

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nt, M., Malmkvist, J., Kaiser, M., Foldager, L., Herskin, M.S. (2022). Piglets’ acute responses to local anesthetic injection and surgical castration: Effects of the injection method and interval between injection and castration. Front. Vet. Sci. 9:1009858. doi: 10.3389/fvets.2022.1009858 7. Dove, C.R., Alworth, L.C. (2015). Blood collection from the orbital sinus of swine. Lab Anim. (NY) 44:383-384. doi: 10.1038/laban.869 . Everaert, N., Van Cruchten, S., Weström, B., Bailey, M., Van Ginneken, C., Thymann, T., Pieper, R. (2017). A review on early gut maturation and colonization in pigs, including biological and dietary factors affecting gut homeostasis. Anim. Feed Sci. Technol. 233:89-103. doi: https://doi.org/10.1016/j.anifeedsci.2017.06.011 9. Fardisi, M., Thelen, K., Groenendal, A., Rajput, M., Sebastian, K., Contreras, G.A., Moeser, A.J. (2023). Early weaning and biological sex shape long-term immune and metabolic responses in pigs. Sci. Rep. 13:15907. doi: https://doi.org/10.1038/s41598-023-42553-9

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.B., Marques, M.N., Cardoso, J.C., Severino, P., Souto, E.B., Albuquerque-Júnior, R.L.C. (2021). Histological evidence of wound healing improvement in rats treated with oral administration of hydroalcoholic extract of Vitis labrusca . Curr. Issues Mol. Biol. 43:335-352. doi: https://doi.org/10.3390/cimb43010028 40. Schmid, S.M., Genter, C.I., Heinemann, C., Steinhoff-Wagner, J. (2021). Impact of tearing spermatic cords during castration in live and dead piglets and consequences on welfare. Porc. Health Manag. 7:17. doi: https://doi.org/10.1186/s40813-021-00200-7 41. Sheil, M.L., Chambers, M., Sharpe, B. (2020). Topical wound anaesthesia: E cacy to mitigate piglet castration pain. Aust. Vet. J. 98:77-83. doi: https://doi.org/10.1111/avj.12930 42. Stewart, M., Verkerk, G.A., Stafford, K.J., Schaefer, A.L., Webster, J.R. (2010). Noninvasive assessment of autonomic activity for evaluation of pain in calves, using surgical castration as a model. J. Dairy Sci. 93:3602-3609. doi: 10.3168/jds.2010-3114 43. Sutherland, M.A., Davis, B.L., Brooks, T.A., McGlone, J.J. (2010). Physiology and behavior of pigs before and after castration: Effects of two topical anesthetics. Animal 4:2071-2079. doi: https://doi.org/10.1017/S1751731110001291 44. Telles, F.G., Luna, S.P.L., Teixeira, G., Berto, D.A. (2016). Long-term weight gain and economic impact in pigs castrated under local anaesthesia. Vet. Anim. Sci. 1:36-39. doi: http://dx.doi.org/10.1016/j.vas.2016.11.003 45. Tridelta Development Ltd. (2024). Tridelta PHASE haptoglobin assay (cat. no. TP-801). Available at: http://www.trideltaltd.com/includes/tng/pub/tNG_download4.php?LINK=haptoglobin-main-

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Page 16 / 20 page&KT_download1=ee32cfdeaf60f2d43cfd4860df9b585d. 4 . van de Vyver, M., Boodhoo, K., Frazier, T., Hamel, K., Kopcewicz, M., Levi, B., Maartens, M., Machcinska, S., Nunez, J., Pagani, C., Rogers, E., Walendzik, K., Wisniewska, J., Gawronska-Kozak, B., Gimble, J.M. (2021). Histology scoring system for murine cutaneous wounds. Stem Cells Dev. 30:1141-1152. doi: https://doi.org/10.1089/scd.2021.0124 47. Viscardi, A.V., Cull, C.A., Kleinhenz, M.D., Montgomery, S., Curtis, A., Lechtenberg, K., Coetzee, J.F. (2020). Using a CO2 surgical laser for piglet castration to reduce pain and in ammation, and to improve wound healing. Kansas Agric. Exp. Stn. Res. Rep. 6:10.. https://doi.org/ 10.4148/2378- 5977.8016 4 . Weiler, U., Font-i-Furnols, M., Tomasevi č , I., Bonneau, M. (2021). Alternatives to piglet castration: From issues to solutions. Animals 11:1041. doi: https://doi.org/10.3390/ani11041041 Table 4 Table 4 is available in the Supplementary Files section. Figures Figure 1 Overview of the experimental timeline and data collection points. Page 17 / 20 Figure 2 Least-squares mean body weight (kg ± SEM) of piglets across treatments and days. Castrated piglets were treated with Iodine (n=32), Oinkment (n=32), PhytoCare (n=31), Vetericyn (n=31), or Zinc Oxide (n=32). The control group consisted of non-castrated (NoCast) piglets treated with iodine (n=32). Statistical analysis showed no effect of treatment ( P > 0.05), a signi cant effect of timepoint ( P < 0.05), and no treatment × timepoint interaction ( P > 0.05). Values are presented as least squares mean ± standard error of the mean (LSM ± SEM).

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< 0.05), and no treatment × timepoint interaction ( P > 0.05). Values are presented as least squares mean ± standard error of the mean (LSM ± SEM). Page 18 / 20 Figure 3 Least-squares mean prostaglandin E ₂ (PGE ₂ ) concentrations (pg/mL ± SEM) in piglets across treatments and days. *Castrated piglets were treated with Iodine (n=32), Oinkment (n=32), PhytoCare (n=31), Vetericyn (n=31), or Zinc Oxide (n=32). The control group consisted of non-castrated (NoCast) piglets treated with iodine (n=32). Statistical analysis showed no effect of treatment ( P > 0.05), a signi cant effect of timepoint ( P < 0.05), and no treatment × timepoint interaction ( P > 0.05). Values are presented as least squares mean ± standard error of the mean (LSM ± SEM). Page 19 / 20 Figure 4 Least-squares mean plasma haptoglobin concentrations (mg/dL ± SEM) in piglets across treatments and timepoints. *Castrated piglets were treated with Iodine (n=32), Oinkment (n=32), PhytoCare (n=31), Vetericyn (n=31), or Zinc Oxide (n=32). The control group consisted of non-castrated (NoCast) piglets treated with iodine (n=32). Statistical analysis showed no effect of treatment ( P > 0.05), a signi cant effect of timepoint ( P < 0.05), and no treatment × timepoint interaction ( P > 0.05). Values are presented as least squares mean ± standard error of the mean (LSM ± SEM).

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0.05), and no treatment × timepoint interaction ( P > 0.05). Values are presented as least squares mean ± standard error of the mean (LSM ± SEM). Page 20 / 20 Figure 5 Least-squares mean skin surface temperature (°C ± SEM) measured via infrared thermography in piglets across treatments and timepoints. *Castrated piglets were treated with Iodine (n=32), Oinkment (n=32), PhytoCare (n=31), Vetericyn (n=31), or Zinc Oxide (n=32). The control group consisted of non-castrated (NoCast) piglets treated with iodine (n=32). Statistical analysis showed an effect of treatment ( P < 0.05), day ( P < 0.05), and the treatment × day interaction ( P < 0.05) Values are presented as least squares mean ± standard error of the mean (LSM ± SEM). Supplementary Files This is a list of supplementary les associated with this preprint. Click to download. SupplemMaterial.docx Table4.docx