TYPE Original Research PUBLISHED 12 September 2022 DOI 10.3389/fvets.2022.936838 Inflammation interferes with OPEN ACCESS chemoreception in pigs by altering the neuronal layout of EDITED BY Michał Dziecioł, Wroclaw University of Environmental and Life Sciences, Poland REVIEWED BY the vomeronasal sensory Archunan Govindaraju, Bharathidasan University, India Valeria Grieco, epithelium University of Milan, Italy *CORRESPONDENCE Violaine Mechin1*, Pietro Asproni1 , Cécile Bienboire-Frosini2 , Violaine Mechin v.mechin@group-irsea.com Alessandro Cozzi3 , Camille Chabaud2 , Sana Arroub4 , SPECIALTY SECTION Eva Mainau5 , Patricia Nagnan-Le Meillour6 and Patrick Pageat3 This article was submitted to 1 Animal Behavior and Welfare, Tissue Biology and Chemical Communication Department, IRSEA, Institute of Research in a section of the journal Semiochemistry and Applied Ethology, Apt, France, 2 Molecular Biology and Chemical Frontiers in Veterinary Science Communication Department, IRSEA, Institute of Research in Semiochemistry and Applied Ethology, Apt, France, 3 Research and Education Board, IRSEA, Institute of Research in Semiochemistry and RECEIVED 05 May 2022 Applied Ethology, Apt, France, 4 Statistics and Data Management Service, IRSEA, Institute of Research ACCEPTED 18 August 2022 in Semiochemistry and Applied Ethology, Apt, France, 5 Department of Animal and Food Science, PUBLISHED 12 September 2022 School of Veterinary Science, Universitat Autònoma de Barcelona, Barcelona, Spain, 6 Univ. Lille, CITATION CNRS, INRAE, UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France Mechin V, Asproni P, Bienboire-Frosini C, Cozzi A, Chabaud C, Arroub S, Mainau E, Nagnan-Le Meillour P and Pageat P Chemical communication is widely used by animals to exchange information (2022) Inflammation interferes with in their environment, through the emission and detection of semiochemicals to chemoreception in pigs by altering the maintain social organization and hierarchical rules in groups. The vomeronasal neuronal layout of the vomeronasal sensory epithelium. organ (VNO) is one of the main detectors of these messages, and its Front. Vet. Sci. 9:936838. inflammation has been linked to behavioral changes because it potentially doi: 10.3389/fvets.2022.936838 prevents molecule detection and, consequently, the translation of the signal COPYRIGHT into action. Our previous study highlighted the link between the intensity © 2022 Mechin, Asproni, Bienboire-Frosini, Cozzi, Chabaud, of vomeronasal sensory epithelium (VNSE) inflammation, probably induced Arroub, Mainau, Nagnan-Le Meillour by farm contaminant exposure, and intraspecific aggression in pigs. The and Pageat. This is an open-access article distributed under the terms of aim of this study was to evaluate the cellular and molecular changes that the Creative Commons Attribution occur during vomeronasalitis in 76 vomeronasal sensorial epithelia from License (CC BY). The use, distribution 38 intensive-farmed pigs. Histology was used to evaluate the condition of or reproduction in other forums is permitted, provided the original each VNO and classify inflammation as healthy, weak, moderate, or strong. author(s) and the copyright owner(s) These data were compared to the thickness of the sensorial epithelium and are credited and that the original publication in this journal is cited, in the number of type 1 vomeronasal receptor cells using anti-Gαi2 protein accordance with accepted academic immunohistochemistry (IHC) and analysis. The presence of odorant-binding practice. No use, distribution or proteins (OBPs) in the areas surrounding the VNO was also analyzed by reproduction is permitted which does not comply with these terms. IHC and compared to inflammation intensity since its role as a molecule transporter to sensory neurons has been well-established. Of the 76 samples, 13 (17%) were healthy, 31 (41%) presented with weak inflammation, and 32 (42%) presented with moderate inflammation. No severe inflammation was observed. Epithelial thickness and the number of Gαi2+ cells were inversely correlated with inflammation intensity (Kruskal–Wallis and ANOVA tests, p < 0.0001), while OBP expression in areas around the VNO was increased in inflamed VNO (Kruskal–Wallis test, p = 0.0094), regardless of intensity. Frontiers in Veterinary Science 01 frontiersin.org Mechin et al. 10.3389/fvets.2022.936838 This study showed that inflammation was associated with a reduction in the thickness of the sensory epithelium and Gαi2+ cell number, suggesting that this condition can induce different degrees of neuronal loss. This finding could explain how vomeronasalitis may prevent the correct functioning of chemical communication, leading to social conflict with a potential negative impact on welfare, which is one of the most important challenges in pig farming. KEYWORDS vomeronasal organ, inflammation, chemodetection, chemical communication, pig Introduction social, maternal, or sexual behaviors in different species (15– 19). Asproni and colleagues revealed that the presence of Chemical communication plays a key role in animal life VNO spontaneous inflammation (vomeronasalitis) was also as it ensures the exchange of semiochemical information associated with intraspecific aggression in cats (20). This link between subjects of the same or different species. This type was recently confirmed in a stable social group of pigs, in which of intraspecific or interspecific communication is performed a strong correlation was found between the histological scores through the exchange of chemical signals in all aspects of VNO inflammation intensity and the number of skin lesions of animal life, including maternal recognition, reproduction, induced by social fighting (21). territorial marking, and predatory/prey recognition (1–3). These In fact, the animal social structure is composed of a set chemosignals are composed of molecules of different nature of behaviors, as in pigs or wild boars, in which a basal level that are released by the emitting animal (through biological of aggression always exists, even in stable social groups, to fluids or secretions, scent marks, etc.) and are detected by ensure hierarchy rules (22). In pigs, signs of aggression are sensory organs, such as the accessory olfactory system, and linked to long-term social stress, which impacts their welfare and especially the vomeronasal organ (VNO) (3–5). Also, the main productivity (23, 24). To date, the link between vomeronasalitis olfactory system is known to attribute excellent olfactory abilities and behavior has been verified (21). to most mammals as in reproduction or in social responses The purpose of the present study was to investigate the (6–8) due to to a large and organized olfactory structure (9, cellular changes that occur in the sensory epithelium of the 10). Concerning the VNO, this tubular and bilateral organ is VNO when inflamed to unveil how vomeronasalitis can alter located in the nasal cavity of most animals and is composed chemical communication capabilities, impacting the behavior of a non-sensory epithelium (NSE) and a sensory epithelium of the affected animals, which could also induce a decrease in (vomeronasal sensory epithelium (VNSE)) arranged around a their welfare. lumen where the environmental air containing semiochemicals The repartition of the three types of receptors transits (11, 12). In most mammals, these molecules are detected (V1Rs, V2Rs, and FPRs) has been proven to be highly by vomeronasal sensory neurons expressing type 1 (V1R) or modified between species (25, 26). In pigs, genomic type 2 (V2R) receptors, distinguishable by their respective analyses detected only the presence of functional V1Rs, coupling with Gαi2 or Gαo proteins in the cytoplasm for further in contrast to V2Rs and FPRs (27). Similar to that in signal transduction (13) and by formyl peptide receptors (FPRs) other species, these results indicate that the V1R gene supposed of acting as chemosensory receptors in the mouse family is responsible for semiochemical detection in VNO (14). pigs (27). The key role of this organ in animal life has been Histology was used to assess the condition of each VNSE ascertained by investigating behavioral modifications after (healthy, weak, or moderate inflammation), which was induced VNO changes, which can provoke alterations in compared to its thickness since these changes have already been shown in olfactory mucosa inflammation (28, 29). Abbreviations: FPR, Formyl Peptide Receptor; Gαi2, guanine Gαi2 protein was studied by immunohistochemistry (IHC) nucleotide-binding protein G (i) subunit alpha-2; Gαo, guanine to identify V1R expressing cells, the main receptor type nucleotide-binding protein G (o) subunit; H&E, Hematoxylin and in pigs. Finally, the expression of the porcine odorant- Eosin; IHC, Immunohistochemistry; OBP, Odorant-Binding Protein; binding protein (OBP) was evaluated by IHC, since NSE, Non-Sensory Epithelium; VNO, Vomeronasal Organ; VNSE, these small soluble proteins secreted in the mucus are Vomeronasal Sensory Epithelium; V1R, Vomeronasal Receptor Type 1; required to permit signal transmission in the receptor V2R, Vomeronasal Receptor Type 2. cells (30, 31). Frontiers in Veterinary Science 02 frontiersin.org Mechin et al. 10.3389/fvets.2022.936838 Materials and methods expressed in micrometers. The VNSE thickness measurement of the five parts follow the scheme illustrated in Figure 1. The VNO samples used in this study were collected from a large project approved by the Institutional Animal Care and Use Committee of the Institute of Agrifood Research and Immunohistochemical analysis Technology (IRTA) and Generalitat de Catalunya (protocol number 7622). After deparaffinization and rehydration, VNO sections were subjected to microwave antigen retrieval in 0.1 M citrate buffer pH 6 solution (Cat No. F/T0050; DiaPath SpA, Martinengo, Animals and sampling procedures Italy) at 560 W for 3 min and 30 s, followed by 15 min at 210 W. Endogenous peroxidase blocking was performed using 100 µl of This study included 76 VNOs sampled from 38 6-months- peroxidase blocking solution (Cat No. ACA500; Scytek, Logan, old female pigs ([Landrace × Large White] × Piétrain), UT, USA) for 30 min. The sections were rinsed and incubated corresponding to a population used for porcine VNO in a for 1 h at room temperature with the primary antibodies. Anti- previous study (21) and to the common age of slaughtering, Gαi2 antibody (rabbit polyclonal, Cat No. sc-7276; Santa Cruz which allows having a representative image of what happens Biotechnologies, Dallas, TX, USA) was diluted at 1:200, and anti- in a farm pig VNO during its productive cycle. They porcine OBP [rabbit polyclonal, provided by Dr P. Nagnan-Le were maintained in the Institute of Agrifood Research and Meillour’s laboratory (30)] was diluted at a ration of 1:10,000. Technology (IRTA, Monells, Spain) facilities in slatted pens (5 IHC anti-Gαi2 protein was performed to identify and count × 2.7 m) with water and food ad libitum. At 23 to 27 weeks V1Rs expressing neurons in the VNSE, and IHC was performed of age (mean 108.0 ± SD 12.4 kg of body weight), they were to identify the presence of OBP in the area surrounding the exposed to a 90% CO2 stunner for 3 min before exsanguination. VNO. The slides were rinsed in Tris-Buffered-Saline (TBS)– Immediately after death, snouts were collected and immersed in Tween 1:100 and incubated with a secondary biotinylated 10% buffered formalin (pH 7.4) until complete tissue fixation. anti-rabbit antibody (Cat No. T/ABE125; UltraTek, ScyTek After their extraction from the nasal cavity, the 76 VNOs Laboratories, Logan, UT, USA) for 10 min at room temperature. were trimmed into 2–3 mm thin sections and dehydrated and Finally, streptavidin-peroxidase (Cat No. 12694067, Invitrogen, paraffin-embedded according to routine histological methods. Carlsbad, CA, USA) was applied to the slides for 10 min, Sections (3.5 µm thick) were cut and dried overnight at 37◦ C and visualization was performed using 3,3’-diaminobenzidine R on SuperFrostPlusTM slides (Cat No. 10149870, Thermo Fisher tetrahydrochloride (ImmPACT DAB Peroxidase Substrate, Scientific, Illkirch, France) before being subjected to histological Cat No. SK4105; Vector Laboratories, Burlingame, CA, USA) and immunohistochemical analyses. and counterstained with hematoxylin for 2 min. The tissues were dehydrated, cleared in xylene, and mounted. As a negative control, the primary antibodies were replaced with non-immune Histopathological analysis rabbit serum. R Slides were observed using the EVOS FL Auto Imaging Each VNO was stained with hematoxylin and eosin (H&E, System (Thermo Fisher Scientific, Illkirch, France) and images BioOptica, Milan, Italy) to classify VNSE inflammation intensity were obtained for further analysis. Concerning the Gαi2 protein R on a scale from 0 to 3 (0 = absence of any sign of investigation, IHC-positive cells were counted with ImageJ inflammation: healthy epithelium, 1 = weak inflammation; software on the total surface of the VNSE and were then 2 = moderate inflammation, 3 = strong inflammation), as converted to a number of positive cells per 1 mm² of VNSE. previously reported (21). Odorant-binding protein (OBP) positivity was obtained R Hematoxylin and eosin (H&E)-stained sections were also using ImageJ software and its color deconvolution plugin to used to measure the VNSE thickness. Microscopical pictures measure stained pixels corresponding to the presence of OBP R were taken with the microscope EVOS FL Auto Imaging in the total VNO section. We obtained a measure expressed System (Thermo Fisher Scientific, Illkirch, France) and its as the percentage of positivity in the entire VNO soft tissue of software and measurements were obtained with the software each sample. Image J (US National Institute of Health, MD, USA) (32) on five different parts of the VNSE, starting from the basement membrane to the top of the knobs, as described in previous Statistical analysis studies focused on mouse olfactory mucosa inflammation (33). These measurements were repeated on three different Data were analyzed using the Statistical Analysis System representative sections of each VNO to obtain a mean value (SAS 9.4 software© 2002–2012; SAS Institute Inc., Cary, NC, (N = 15), which is considered the definitive VNSE thickness, USA). The significance threshold was set at 5%. Frontiers in Veterinary Science 03 frontiersin.org Mechin et al. 10.3389/fvets.2022.936838 FIGURE 1 Vomeronasal sensory epithelium thickness measurement. Five measures (red crosses) were taken by VNSE. (Hematoxylin and eosin (H&E) staining, Objective x10, Scale bar 100 µm). Each VNO was analyzed individually and classified Results according to the VNSE inflammation score. VNSE thickness, Gαi2, and OBP protein expression were independently analyzed Of the 76 VNO samples from 38 intensive-farmed pigs, according to the inflammation score. First, the normality of 13 (17%) were healthy, 31 (41%) presented with weak each parameter was verified using the UNIVARIATE procedure, inflammation, and 32 (42%) had moderate inflammation. No and second, homoscedasticity was checked using the Global severe inflammation was observed. An inflammatory infiltrate Linear Model (GLM) procedure. composed of small lymphocytes was observed in the soft Concerning the VNSE thickness, normality and tissue above the sensory epithelium. Small quantities of homoscedasticity were not verified; therefore, a non-parametric plasma cells and macrophages, such as rare mast cells and alternative Kruskal–Wallis test was used with the NPAR1WAY non-degenerate neutrophils, were present. Neutrophils were procedure. Multiple comparisons were obtained by computing exclusively located in the epithelium. Moderate inflammation the Wilcoxon tests for each pair of modalities of VNSE alteration presented as inflammatory cells infiltrating the vomeronasal scores. Bonferroni correction was applied using the MULTTEST nerves and glands. The descriptive data for all the parameters procedure to control for type I errors. are shown in Table 1. For the Gαi2 protein, normality and homoscedasticity were verified, and conditions were satisfied by applying a one- way ANOVA with the GLM procedure. Multiple comparisons were performed using Tukey–Kramer adjustment by adding Thickness of the epithelium the LS MEANS statement to the procedure. The correlation between the Gαi2 protein levels and VNSE thickness was The thickness of the sensory epithelium was compared with explored using the CORR procedure. As normality was not the degree of inflammation. A significant effect of the VNSE verified for these parameters, a Spearman coefficient (Rhô) inflammation score was observed (DF = 2; χ² = 31.39; p < was used to measure the possible correlation that may exist 0.0001; Kruskal–Wallis test). The multiple comparisons after the between them. With regard to the OBP parameter, normality Bonferroni correction showed a significant decrease in thickness and homoscedasticity were not verified. Consequently, the non- when the VNSE inflammation intensity was increased, between parametric alternative Kruskal–Wallis test was used, followed by those that were healthy and those that were weakly inflamed (p the Wilcoxon two-sample test with a Bonferroni correction for = 0.0003); between those that were healthy and those that were multiple comparisons. moderately inflamed (p = 0.0003); and between those that were Frontiers in Veterinary Science 04 frontiersin.org Mechin et al. 10.3389/fvets.2022.936838 TABLE 1 Mean, standard deviation (STD DEV), standard error (SE), and median (MED) of epithelium thickness, Gαi2+ cells, porcine odorant binding protein (OBP) according to the vomeronasal organ (VNO) inflammation score as 0 = absence; 1 = weak; 2 = moderate; 3 = strong inflammation. VNO inflammation N MEAN STD DEV SE MED Epithelium thickness (µm) 0 13 93.30 5.91 1.64 91.85 1 31 74.16 12.87 2.31 73.01 2 32 65.22 11.14 1.97 61.32 Gαi2+ cells (nb/mm²) 0 13 802.38 98.01 27.18 796.00 1 31 485.65 107.95 19.39 477.00 2 32 358.00 145.45 27.71 347.50 OBP (% positivity) 0 13 0.95 0.37 0.10 0.87 1 31 7.02 7.07 1.27 5.39 2 32 6.44 6.36 1.12 3.94 FIGURE 2 Vomeronasal sensory epithelium (VNSE) thickness decreases with inflammation intensity. Hematoxylin and eosin (H&E) staining was used to measure the thickness. (A) VNSE thickness according to the vomeronasal organ inflammation. Data are expressed in µm and shown as the mean ± SD (*** = p < 0.001; **: p < 0.01). (B) Healthy epithelium, score = 0, (C) Weak inflammation, score =1: few lymphocytes were found under the epithelium (black arrows). (D) Moderate inflammation, score =2: denser inflammatory infiltrate mainly composed of lymphocytes was found above the VNSE (black arrows). (Objective x20, Scale bars 200 µm). Frontiers in Veterinary Science 05 frontiersin.org Mechin et al. 10.3389/fvets.2022.936838 weakly and those that were moderately inflamed (p = 0.0033) that vomeronasalitis strongly impacts the VNO epithelium (Figure 2). structure, inducing important cellular changes. Due to the limited information available concerning the analysis of VNO inflammation and because this organ looks Quantity of Gαi2+ cells like the olfactory mucosa from a histological point of view (2, 3), we compared our results with the literature on olfactory Statistical analyses showed that, when the epithelium was epithelium inflammation (28, 34), which has been more widely inflamed, the number of Gαi2+ cells was significantly reduced studied and found to be in agreement. In fact, our results (DF = 2; Fisher Test (F) = 59.46; p < 0.0001; one-way ANOVA). showed that inflammation of the VNSE induces a reduction in Multiple comparisons revealed a significant difference between its thickness according to inflammation intensity, confirming the VNSE scores of 0 and 1, between the VNSE scores of 0 and what was reported by other studies that showed an association 2 (p < 0.0001 each), and between the scores of 1 and 2 (p = between the reduction of the olfactory epithelium thickness and 0.0003), as presented in Figure 3. its inflammation (29, 39). The inflammatory microenvironment A moderate positive correlation between the thickness and composition has been shown to induce apoptosis (40–42) and quantity of Gαi2 protein expression was observed (rho = 0.62, p olfactory mucosa epithelial thickness reduction (34, 43) and < 0.0001; Spearman’s test). could be an aspect that will deserve to be explored to better understand our results. To verify the impact of vomeronasalitis on neuronal layout, Odorant-binding protein V1R neurons were analyzed immunohistologically. In the VNSE, it is known that the Gαi2 protein plays a major role in Concerning the presence of OBP in the area surrounding communication because it interacts with the V1R in charge of VNSE, protein expression was significantly increased when the detection of small organic/volatile molecules. These signals VNSE was inflamed (DF = 2; χ² = 9.34; p = 0.0094; Kruskal– are used in social communication, as in maternal or sexual Wallis test). Multiple comparisons (with Wilcoxon two-sample exchanges, and provide signal transduction, leading to neuronal tests) indicated that a difference was obtained between grade 0 responses following V1R activation (44–47). Their crucial role and the presence of inflammation in grades 1 and 2 (p = 0.0135 is even more important to be explored in this species, since each), with no difference between grade 1 and grade 2 (p = this kind of receptor is, to date, the only one found in pig 0.9616) (Figure 4). VNSE. In fact, V2Rs, typically characterized in other species such as rodents and marsupials, coupled with the Gαo protein, have never been observed in pigs and in most ungulates and Discussion carnivores (26, 27, 48–51). Diminution or inhibition of Gαi2 gene expression has been Epithelium inflammation has been studied in the human and shown to induce behavioral complications in Gαi2 mutant mice, mouse olfactory epithelium and has been shown to induce a such as modified sexual behaviors or an increase in maternal reduction in epithelium thickness and alterations in olfactory aggression (47, 52). In this study, we found that, when inflamed, capabilities, such as hyposmia and anosmia (28, 29, 34). VNSE possesses fewer cells expressing the Gαi2 protein, clearly It occurs because of many factors, including environmental suggesting a decrease in the number of V1R neurons in VNSE. contaminants such as organic dust or natural pollutant gases In addition to functional organization, the presence of such as ammonia or hydrogen sulfide (35, 36). Since the OBPs in the VNO has been proven essential to ensuring sensory epithelium of the VNO is similar to the olfactory semiochemical detection (30, 53–55). These proteins are epithelium from a morphological and functional point of view secreted by the olfactory epithelium glands in high quantities (2), we can suppose that the pollutants that induce rhinitis in the nasal mucus (56, 57) and are hypothesized to be and olfactory mucosa inflammation induce inflammation of the odorant transporters that deliver these olfactory molecules to VNO. Furthermore, it is well-known that the concentration receptors in pigs (58, 59), cows (53), and other mammals of these environmental contaminants increases under intensive (60, 61). The present study showed that, when inflammation farm conditions because of the high animal density (37, 38). was detected in VNSE, regardless of the intensity, the However, the impact of environmental contaminants on the expression of OBP in the surrounding areas was increased. occurrence of vomeronasalitis requires further investigation. This observation could be explained by the modulatory role Vomeronasalitis has been described in cats and recently of OBP in the inflammatory response proposed by Mitchell in pigs, and in both cases, it was associated with intraspecific et al. (62), as this protein seems to inhibit neutrophil aggressive behavior (20, 21). However, there is a lack of data recruitment by inflammatory mediators in the respiratory on the cellular and molecular characterization of this pathology. system (62). Some studies on other species demonstrated OBP Our study provides more information on this topic, indicating compensatory properties on olfactory systems, where different Frontiers in Veterinary Science 06 frontiersin.org Mechin et al. 10.3389/fvets.2022.936838 FIGURE 3 Gαi2+ cells number decrease with inflammation intensity. Immunohistochemical staining was used to reveal the presence of Gαi2 protein (brown staining shown by arrows). (A) Mean and standard error of the Gαi2 protein according to the vomeronasal organ inflammation. Data are expressed in number of Gαi2+ cells /mm2 and shown as the mean ± SE (*** = p < 0.001). (B) Healthy epithelium, score = 0, (C) Weak inflammation, score =1, (D) Moderate inflammation, score =2. (Objective x40, Scale bars 100 µm). OBP subtypes have been reported to occur simultaneously Under intensive conditions, the environmental air (63–65). These kinds of studies should be exploited in the composition is harmful to the respiratory tract (37, 38) pig species to further analyze the effect of the sensory and, thus, potentially to their VNOs. Intraspecific chemical epithelium damages on the OBP expression and thus on the communication is fundamental in animal life, particularly detection capabilities. in farm animals, since these animals need to use their This study allowed us to better characterize vomeronasalitis communicative skills to better deal with restricted areas and in pigs and the modifications the condition induces in the to exchange with other animals. This study highlights the VNO. Asproni et al. (21) demonstrated that, when inflammation importance of the effects of vomeronasalitis in farm animals, was present in a pig’s VNSE, the animal was more susceptible and it can open novel perspectives focused on the limitation of to aggression by congeners. Our results showed that inflamed its onset to improve welfare, which is strongly linked to animal VNSE possesses a lower number of neurons responsible for behavior and communication. chemoreception. As already shown in the olfactory epithelium, a decreasing number of neurons induces a loss of efficiency such as hyposmia or anosmia (28, 34), driving the authors Conclusion to suppose that the VNO could also be functionally impacted by this loss of neurons, inducing troubles in pig chemical Our results permit the investigation of the molecular and communication, which is crucial in this species due to its cellular mechanisms by which inflammation of the VNO alters social organization. chemodetection in pigs, potentially contributing to the onset Frontiers in Veterinary Science 07 frontiersin.org Mechin et al. 10.3389/fvets.2022.936838 FIGURE 4 Odorant-binding protein (OBP) increases with the presence of inflammation in the vomeronasal sensory epithelium. Immunohistochemical staining was used to reveal the presence of OBP (brown staining shown by arrows). (A) Mean of the OBP protein according to the VNO inflammation. Data are expressed in percentages of OBP expression positivity and shown as the mean ± SD (* = p < 0.05). (B) Healthy epithelium, score = 0, (C) Weak inflammation, score =1, (D) Moderate inflammation, score = 2. (Objective x10, Scale bar 400 µm). of aggressive behaviors in the farm pen. In fact, neuronal Ethics statement loss caused by the inflammatory process seems to critically reduce the chemoreceptive capabilities of the affected animals by The animal study was reviewed and approved by diminishing the VNSE thickness and decreasing the number of Institutional Animal Care and Use Committee of IRTA V1R neurons. To the best of our knowledge, this study is the first and Generalitat de Catalunya. to characterize the effects of vomeronasalitis on VNO function, paving the way for further in-depth studies on the link between chemoreception, animal pathology, behavior, and welfare. Author contributions This study provides new insights into the characterization of Conceptualization: PA, CB-F, PP, and AC. Methodology: VNO inflammation and the mechanisms by which it interferes VM, PA, CC, EM, and PN-LM. Validation, writing—original with chemoreception and animal behavior. draft preparation, and visualization: VM and PA. Formal analysis and data curation: SA. Investigation: VM, PA, CC, and Data availability statement EM. Resources and funding acquisition: PP. Writing—review and editing: PA, CB-F, AC, PN-LM, PP, and SA. Supervision and The raw data supporting the conclusions of this article will project administration: PA. All authors have read and agreed to be made available by the authors, without undue reservation. the published version of the manuscript. Frontiers in Veterinary Science 08 frontiersin.org Mechin et al. 10.3389/fvets.2022.936838 Acknowledgments that could be construed as a potential conflict of interest. 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