CYP2A

…Boar carcasses can have higher skatole levels in adipose tissue than barrows or gilts because the hepatic degradation of skatole is reduced, due to lower activities of CYP2E1 and CYP2A enzymes if…

…Boar carcasses can have higher skatole levels in adipose tissue than barrows or gilts because the hepatic degradation of skatole is reduced, due to lower activities of CYP2E1 and CYP2A enzymes if…

oky and phenolic nuances. The detection of heptyl mercaptan and propylene glycol further introduced sulphurous and alcoholic components to the overall aroma pro- file. In contrast, the E2 group was characterised by the presence of 2,3-butanediol, octane, p-menthatriene, and (E,E)-2,4-nonadienal, associated with bitter, woody, and cereal odour notes. These compounds contributed to a more balanced and mild overall aroma profile compared with the other groups. Overall, the PCA separated immunocastrated and un- castrated boars primarily along PC1, reflecting a clear transition from terpene-dominated profiles, typical of uncastrated boars, toward aldehyde and ester profiles characteristic of immunocastrated pigs. The PCA biplot (Figure 2) shows the distribution of backfat samples in a two- dimensional space defined by PC1 (23.55%) and PC2 (15.76%), jointly explaining 39.31% of the total variance. Distinct groupings of the samples are visible, with partial overlap between groups C and E1, and clearer separation from group E2. The spatial configuration of the groups reflects the variability in the volatile compound profile of the backfat. Fat obtained from boars from the control group (C) was characterised by the presence of al- cohols (1-propanol, 2-octanol) and terpene (terpinolene), as well as aldehyde (but-2-enal). These compounds contributed to alcoholic, fatty, green, and anisic odour notes, which are Animals 2025, 15, 3374 12 of 19 commonly associated with the lipid oxidation processes. The E1 group exhibited unique volatile components, including formic acid and hexanoic acid (carboxylic acids with pun- gent and fatty notes), 1-butanamine (amine with a fishy odour), and sotolon (a lactone associated with mushroom aroma). This combination indicates the presence of

oky and phenolic nuances. The detection of heptyl mercaptan and propylene glycol further introduced sulphurous and alcoholic components to the overall aroma pro- file. In contrast, the E2 group was characterised by the presence of 2,3-butanediol, octane, p-menthatriene, and (E,E)-2,4-nonadienal, associated with bitter, woody, and cereal odour notes. These compounds contributed to a more balanced and mild overall aroma profile compared with the other groups. Overall, the PCA separated immunocastrated and un- castrated boars primarily along PC1, reflecting a clear transition from terpene-dominated profiles, typical of uncastrated boars, toward aldehyde and ester profiles characteristic of immunocastrated pigs. The PCA biplot (Figure 2) shows the distribution of backfat samples in a two- dimensional space defined by PC1 (23.55%) and PC2 (15.76%), jointly explaining 39.31% of the total variance. Distinct groupings of the samples are visible, with partial overlap between groups C and E1, and clearer separation from group E2. The spatial configuration of the groups reflects the variability in the volatile compound profile of the backfat. Fat obtained from boars from the control group (C) was characterised by the presence of al- cohols (1-propanol, 2-octanol) and terpene (terpinolene), as well as aldehyde (but-2-enal). These compounds contributed to alcoholic, fatty, green, and anisic odour notes, which are Animals 2025, 15, 3374 12 of 19 commonly associated with the lipid oxidation processes. The E1 group exhibited unique volatile components, including formic acid and hexanoic acid (carboxylic acids with pun- gent and fatty notes), 1-butanamine (amine with a fishy odour), and sotolon (a lactone associated with mushroom aroma). This combination indicates the presence of

before mum likelihood estimation linear mixed model analy- animal motor response and withdrawal from the device. ses were used to analyze differences between treatment von Frey Monofilament. Von Frey monofilaments groups for all behavioral and wound sensitivity testing are calibrated to bend at a predetermined pressure to data, with the factors time, treatment, and interaction be- provide repeatable pain stimulation of predetermined tween time and treatment as explanatory variables and sites on the wound and surrounding (periwound) skin, calf number as the random effect. Where a significant as previously described in lambs (Lomax et al., 2008, interaction was found, post hoc pairwise comparisons 2010). A 300-g filament was used to perform direct sen- using LSD were performed to analyze between-group sory testing at 4 predetermined sites on the cut skin edge differences (Table 1). For all statistical calculations, P < of the scrotal wound and 2 sites on the intact skin sur- 0.05 was considered statistically significant. rounding the wound. Evidence of local anesthesia (diminished response RESULTS to tactile stimulation) and primary and secondary hy- peralgesia (heightened response to stimulus directly in Pain-Related Behavior the damaged tissue or in surrounding undamaged tis- sue, respectively) were assessed at each site. Responses There was a significant treatment effect on pain- were scored by monitoring induced motor reflexes in the related behavior (Table 2). Calves treated with TA ex- 4948 Lomax and Windsor Table 1. Statistical interactions and df tested in Trials 1 Response scores in the TA-treated calves were sig- and 2 nificantly below those of the untreated calves between 2 Response Wald and 24 h after castration (P < 0.001; Table 4). Response Trial variate

lable, pre-treatment data were included as a co-variate. Baseline data were defined as the latest measurement of a given variable before treatment was administered. The statistical model included treatment, sex (except for parameters only measured in one sex), and treatment-by-sex interactions as fixed effects, while the repeated measures ANOVA model included treatment, sex and time (and their 2-way and 3-way interac- tions) as fixed effects. Data were compared either within or across sex depending on observed interactions. Models were selected using backwards elimination where 3-way and 2-way interaction terms with the highest p-Value were sequentially dropped from the model. The process was performed hierarchically starting with the 3-way interaction terms using a threshold p-Value of 0.1, as required by regulatory guidelines. Model selection was stopped when any of the following occurred: (i) all interaction terms at the highest level had a p-Value < 0.1; (ii) no interaction terms remained in the model. The statistical model was therefore: Parameter = Baseline_variables + Treatment + Sex + Time + Treatment:Sex + Treat- ment:Time + Sex:Time + Treatment:Sex:Time + Residual_Error Least squares means were compared at a significance level of p < 0.1 in the first instance apart from treatment by sex by time point, which was compared at p < 0.05 in the first instance. For the repeated measures models, most residual error patterns were selected based on a comparison of Akaike Information Criteria (AIC) output for models containing an unstructured (UN), autoregressive (AR; 1), variance components (VC) and independent residual error pattern (with the lowest AIC preferred among the possible covariance matrices). The denominator degrees of freedom in the covariance pattern

oler 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 Scientific, Corning, NY) at − 80°C. The assays for the biomarker analysis were completed six to nine months post-collection. PGE2 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). Briefly, serum samples were purified 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 PGE2 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 coefficient 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 Page 5/20 assay procedure involved

oler 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 Scientific, Corning, NY) at − 80°C. The assays for the biomarker analysis were completed six to nine months post-collection. PGE2 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). Briefly, serum samples were purified 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 PGE2 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 coefficient 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 Page 5/20 assay procedure involved

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tor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.