Chemical communication plays a key role in animal life as it ensures the exchange of semiochemical information between subjects of the same or different species. This type of intraspecific or interspecific communication is performed through the exchange of chemical signals in all aspects of animal life, including maternal recognition, reproduction, territorial marking, and predatory/prey recognition (1–3). These chemosignals are composed of molecules of different nature that are released by the emitting animal (through biological fluids or secretions, scent marks, etc.) and are detected by sensory organs, such as the accessory olfactory system, and especially the vomeronasal organ (VNO) (3–5). Also, the main olfactory system is known to attribute excellent olfactory abilities to most mammals as in reproduction or in social responses (6–8) due to to a large and organized olfactory structure (9, 10). Concerning the VNO, this tubular and bilateral organ is located in the nasal cavity of most animals and is composed of a non-sensory epithelium (NSE) and a sensory epithelium (vomeronasal sensory epithelium (VNSE)) arranged around a lumen where the environmental air containing semiochemicals transits (11, 12). In most mammals, these molecules are detected by vomeronasal sensory neurons expressing type 1 (V1R) or type 2 (V2R) receptors, distinguishable by their respective coupling with Gαi2 or Gαo proteins in the cytoplasm for further signal transduction (13) and by formyl peptide receptors (FPRs) supposed of acting as chemosensory receptors in the mouse VNO (14).

The key role of this organ in animal life has been ascertained by investigating behavioral modifications after induced VNO changes, which can provoke alterations in social, maternal, or sexual behaviors in different species (15–19). Asproni and colleagues revealed that the presence of VNO spontaneous inflammation (vomeronasalitis) was also associated with intraspecific aggression in cats (20). This link was recently confirmed in a stable social group of pigs, in which a strong correlation was found between the histological scores of VNO inflammation intensity and the number of skin lesions induced by social fighting (21).

In fact, the animal social structure is composed of a set of behaviors, as in pigs or wild boars, in which a basal level of aggression always exists, even in stable social groups, to ensure hierarchy rules (22). In pigs, signs of aggression are linked to long-term social stress, which impacts their welfare and productivity (23, 24). To date, the link between vomeronasalitis and behavior has been verified (21).

The purpose of the present study was to investigate the cellular changes that occur in the sensory epithelium of the VNO when inflamed to unveil how vomeronasalitis can alter chemical communication capabilities, impacting the behavior of the affected animals, which could also induce a decrease in their welfare.

The repartition of the three types of receptors (V1Rs, V2Rs, and FPRs) has been proven to be highly modified between species (25, 26). In pigs, genomic analyses detected only the presence of functional V1Rs, in contrast to V2Rs and FPRs (27). Similar to that in other species, these results indicate that the V1R gene family is responsible for semiochemical detection in pigs (27).

Histology was used to assess the condition of each VNSE (healthy, weak, or moderate inflammation), which was compared to its thickness since these changes have already been shown in olfactory mucosa inflammation (28, 29). Gαi2 protein was studied by immunohistochemistry (IHC) to identify V1R expressing cells, the main receptor type in pigs. Finally, the expression of the porcine odorant-binding protein (OBP) was evaluated by IHC, since these small soluble proteins secreted in the mucus are required to permit signal transmission in the receptor cells (30, 31).

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 Technology (IRTA) and Generalitat de Catalunya (protocol number 7622).

Of the 76 VNO samples from 38 intensive-farmed pigs, 13 (17%) were healthy, 31 (41%) presented with weak inflammation, and 32 (42%) had moderate inflammation. No severe inflammation was observed. An inflammatory infiltrate composed of small lymphocytes was observed in the soft tissue above the sensory epithelium. Small quantities of plasma cells and macrophages, such as rare mast cells and non-degenerate neutrophils, were present. Neutrophils were exclusively located in the epithelium. Moderate inflammation presented as inflammatory cells infiltrating the vomeronasal nerves and glands. The descriptive data for all the parameters are shown in Table 1.

Epithelium inflammation has been studied in the human and mouse olfactory epithelium and has been shown to induce a reduction in epithelium thickness and alterations in olfactory capabilities, such as hyposmia and anosmia (28, 29, 34). It occurs because of many factors, including environmental contaminants such as organic dust or natural pollutant gases such as ammonia or hydrogen sulfide (35, 36). Since the sensory epithelium of the VNO is similar to the olfactory epithelium from a morphological and functional point of view (2), we can suppose that the pollutants that induce rhinitis and olfactory mucosa inflammation induce inflammation of the VNO. Furthermore, it is well-known that the concentration of these environmental contaminants increases under intensive farm conditions because of the high animal density (37, 38). However, the impact of environmental contaminants on the occurrence of vomeronasalitis requires further investigation.

Vomeronasalitis has been described in cats and recently in pigs, and in both cases, it was associated with intraspecific aggressive behavior (20, 21). However, there is a lack of data on the cellular and molecular characterization of this pathology. Our study provides more information on this topic, indicating that vomeronasalitis strongly impacts the VNO epithelium structure, inducing important cellular changes.

Due to the limited information available concerning the analysis of VNO inflammation and because this organ looks like the olfactory mucosa from a histological point of view (2, 3), we compared our results with the literature on olfactory epithelium inflammation (28, 34), which has been more widely studied and found to be in agreement. In fact, our results showed that inflammation of the VNSE induces a reduction in its thickness according to inflammation intensity, confirming what was reported by other studies that showed an association between the reduction of the olfactory epithelium thickness and its inflammation (29, 39). The inflammatory microenvironment composition has been shown to induce apoptosis (40–42) and olfactory mucosa epithelial thickness reduction (34, 43) and could be an aspect that will deserve to be explored to better understand our results.

To verify the impact of vomeronasalitis on neuronal layout, V1R neurons were analyzed immunohistologically. In the VNSE, it is known that the Gαi2 protein plays a major role in communication because it interacts with the V1R in charge of the detection of small organic/volatile molecules. These signals are used in social communication, as in maternal or sexual exchanges, and provide signal transduction, leading to neuronal responses following V1R activation (44–47). Their crucial role is even more important to be explored in this species, since this kind of receptor is, to date, the only one found in pig 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 carnivores (26, 27, 48–51).

Diminution or inhibition of Gαi2 gene expression has been shown to induce behavioral complications in Gαi2 mutant mice, such as modified sexual behaviors or an increase in maternal aggression (47, 52). In this study, we found that, when inflamed, VNSE possesses fewer cells expressing the Gαi2 protein, clearly suggesting a decrease in the number of V1R neurons in VNSE.

In addition to functional organization, the presence of OBPs in the VNO has been proven essential to ensuring semiochemical detection (30, 53–55). These proteins are secreted by the olfactory epithelium glands in high quantities in the nasal mucus (56, 57) and are hypothesized to be odorant transporters that deliver these olfactory molecules to receptors in pigs (58, 59), cows (53), and other mammals (60, 61). The present study showed that, when inflammation was detected in VNSE, regardless of the intensity, the expression of OBP in the surrounding areas was increased. This observation could be explained by the modulatory role of OBP in the inflammatory response proposed by Mitchell et al. (62), as this protein seems to inhibit neutrophil recruitment by inflammatory mediators in the respiratory system (62). Some studies on other species demonstrated OBP compensatory properties on olfactory systems, where different OBP subtypes have been reported to occur simultaneously (63–65). These kinds of studies should be exploited in the pig species to further analyze the effect of the sensory epithelium damages on the OBP expression and thus on the detection capabilities.

This study allowed us to better characterize vomeronasalitis in pigs and the modifications the condition induces in the VNO. Asproni et al. (21) demonstrated that, when inflammation was present in a pig's VNSE, the animal was more susceptible to aggression by congeners. Our results showed that inflamed VNSE possesses a lower number of neurons responsible for 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 to suppose that the VNO could also be functionally impacted by this loss of neurons, inducing troubles in pig chemical communication, which is crucial in this species due to its social organization.

Under intensive conditions, the environmental air composition is harmful to the respiratory tract (37, 38) and, thus, potentially to their VNOs. Intraspecific chemical communication is fundamental in animal life, particularly in farm animals, since these animals need to use their communicative skills to better deal with restricted areas and to exchange with other animals. This study highlights the importance of the effects of vomeronasalitis in farm animals, and it can open novel perspectives focused on the limitation of its onset to improve welfare, which is strongly linked to animal behavior and communication.

Our results permit the investigation of the molecular and cellular mechanisms by which inflammation of the VNO alters chemodetection in pigs, potentially contributing to the onset of aggressive behaviors in the farm pen. In fact, neuronal loss caused by the inflammatory process seems to critically reduce the chemoreceptive capabilities of the affected animals by diminishing the VNSE thickness and decreasing the number of V1R neurons. To the best of our knowledge, this study is the first 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.

This study provides new insights into the characterization of VNO inflammation and the mechanisms by which it interferes with chemoreception and animal behavior.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Institutional Animal Care and Use Committee of IRTA and Generalitat de Catalunya.

Conceptualization: PA, CB-F, PP, and AC. Methodology: VM, PA, CC, EM, and PN-LM. Validation, writing—original draft preparation, and visualization: VM and PA. Formal analysis and data curation: SA. Investigation: VM, PA, CC, and EM. Resources and funding acquisition: PP. Writing—review and editing: PA, CB-F, AC, PN-LM, PP, and SA. Supervision and project administration: PA. All authors have read and agreed to the published version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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