Immunolocalization and immunoexpression levels of the novel peptide phoenixin-14 and its receptor GPR173 in the gastrointestinal tract of calves and adult domestic cattle (Bos taurus taurus)

Immunolocalization and immunoexpression levels of the novel peptide phoenixin-14 and its receptor GPR173 in the gastrointestinal tract of calves and adult domestic cattle (Bos taurus taurus) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Immunolocalization and immunoexpression levels of the novel peptide phoenixin-14 and its receptor GPR173 in the gastrointestinal tract of calves and adult domestic cattle (Bos taurus taurus) Katarzyna Kras, Cezary Osiak-Wicha, Marcin B. Arciszewski This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4852060/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Feb, 2025 Read the published version in BMC Veterinary Research → Version 1 posted 13 You are reading this latest preprint version Abstract Phoenixin (PNX), an ancient but newly discovered neuropeptide, is involved in various physiological processes, such as food intake, cardiovascular functions, reproductive functions, and stress regulation. The peptide is derived from the precursor protein small integral membrane protein 20 (SMIM20) and acts through the GPR173 receptor. Due to its relatively recent discovery in 2013, there is a gap in research regarding its localization in specific organs. There are no data in the literature concerning its location and level in the gastrointestinal tract (GIT) of domestic cattle, which are among the world's main livestock animals. Due to the fact that PNX exhibits a highly conserved structure across species, it is likely that it performs key functions in the body. Therefore, this study aimed to investigate the immunolocalization and immunoexpression levels of PNX-14 and GPR173 in the GIT segments of calves and adult cattle. Study material, including GIT sections of two age groups, adults and calves of domestic cattle (n = 6), was obtained from a slaughterhouse. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemical (IHC) analyses were performed. Analyses revealed low levels of PNX-14 in the GIT of both age groups, with localization restricted to epithelial cells across all examined GIT segments. The highest levels were observed in the rumen and reticulum, higher in adults than in calves, whereas the levels in the abomasum and intestines were higher in calves than in adults. This distribution may result from the delayed development of forestomachs in calves. The higher level of GPR173 than PNX-14 and its broader distribution may suggest that PNX-14 is not the only ligand for this receptor. Overall, the results suggest that both peptides may play protective roles related to the immune response, regulate digestive and absorptive functions, and due to receptor presence in nerve fibres, may play a role in regulating GIT secretion and motility. These findings could potentially facilitate further research into the therapeutic potential of targeting PNX-14 and GPR173 in managing gastrointestinal disorders in domestic cattle and other species. cattle developmental metabolism GPCR PNX-14 polygastric animals ruminants SMIM20 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Phoenixin (PNX), although probably one of the oldest discovered neuropeptides, has just recently been identified and has sparked significant interest because of its wide-ranging physiological roles and highly conserved structure across species ( 1 , 2 ). PNX is derived from the enzymatic cleavage of small integral membrane protein 20 (SMIM20), a precursor protein encoded by the SMIM20 gene. This cleavage results in the production of two primary isoforms: PNX-14, which is 14 amino acids long, and PNX-20, which consists of 20 amino acids ( 2 , 3 ). SMIM20 mRNA expression has been extensively investigated in both central and peripheral tissues, including the gastrointestinal tract (GIT), heart, and lungs. Notably, the highest expression levels were found in the hypothalamus, followed by the heart, with lower levels detected in other tissues ( 2 , 4 ). The detection of PNX in various species, such as humans, rodents, pigs, cattle, and chickens, coupled with its highly conserved structure, particularly in mammals, suggests a crucial biological role for this peptide ( 5 ). Discovered in 2013 by Yosten et al. through a bioinformatic approach ( 2 ), PNX has been implicated in various regulatory functions within the body. This peptide is particularly notable for its involvement in critical processes such as food intake, reproductive function, and cardiovascular regulation ( 5 , 6 ). As research into PNX progresses, its potential impact on different bodily systems is becoming more evident, with emerging studies highlighting its roles in mental health, including anxiety and depression ( 7 ). Nevertheless, the functional repertoire of PNX is expanding as new roles are continuously uncovered. However, the role of PNX in the GIT remains elusive due to the scarcity of related research. Preliminary findings suggest a protective role for PNX against duodenal ulcers ( 8 ). Notably, the regulation of energy balance, including food intake, is intrinsically linked to reproductive functions and is mediated through the hypothalamic‒pituitary‒gonadal (HPG) axis. This axis, in turn, is also connected with anxiety and depression, highlighting the multifaceted interactions among these processes ( 9 ). PNX exerts its effects primarily through the GPR173 receptor, also known as the super-conserved receptor expressed in the brain 3 (SREB3), which is part of the extensive family of G protein-coupled receptors (GPCRs). Localization studies of GPR173 have confirmed its presence in the hypothalamus, pituitary gland, and ovaries, supporting its association with the HPG axis ( 5 , 7 ). Additionally, GPR173 is expressed in the heart, skin, and pancreas ( 10 ). Recent findings suggest a dual role for GPR173, not only as a receptor for PNX but also as a potential receptor for cholecystokinin (CCK), although this remains a point of contention ( 11 ). Despite the detection of SMIM20 and GPR173 mRNA across all segments of the GIT in both calves and adult cattle ( 12 ), there is a notable gap in our understanding of the actual presence and immunolocalization of the PNX and GPR173 proteins in these tissues. This study aims to fill this gap by investigating the immunolocalization and immunoexpression levels of PNX-14 and GPR173 in distinct segments of the GIT of calves and adult cattle. Given the observed presence of SMIM20 mRNA throughout the GIT in ruminants, we hypothesize that PNX-14 and its receptor GPR173 are immunolocalized in distinct patterns across different segments of the GIT in these animals. We further propose that the levels of immunoexpression of these proteins vary between calves and adult cattle, reflecting the developmental differences in metabolic activity and gastrointestinal function. Specifically, we expect higher immunoexpression levels in segments of the GIT that are more metabolically active or structurally complex, such as the rumen in adults and the abomasum in calves. This differential expression may indicate specialized roles for PNX-14 and GPR173 in regulating the physiological processes of digestion and nutrient absorption during different stages of development. Understanding these patterns could provide valuable insights into the functional roles of PNX-14 and GPR173 in ruminant physiology and inform strategies for optimizing nutrition and health in livestock. Results PNX and GPR173 immunolocalization Forestomach: rumen, reticulum, and omasum In the forestomach compartments, both PNX-14 and GPR173 exhibited specific and distinct localization patterns. PNX immunoreactivity (IR) was restricted to the mucosal epithelium in all three compartments. In the rumen, reticulum, and omasum, PNX was observed in the cytoplasm of epithelial cells (Fig. 1 A-C). For GPR173, the receptor was present in the mucosal epithelium as granular structures occupying significant areas of the cytoplasm and single nuclei in all of the forestomach compartments (Fig. 2A 1 , B 1 and C 1 ). GPR173 was also detected in the blood vessels (Fig. 2 A 2 , B 2 and C 2 ). Here, it appeared as dispersed granules forming occasional fibre-like arrangements within the vessel walls, with variable intensities across different vessels. Notably, the rumen lacked receptor localization in endothelial cells, in contrast with the reticulum and omasum. Moreover, IR was not observed in every vessel (Fig. 2A 2 ). Abomasum In the abomasum, PNX-IR was primarily observed in the cytoplasm and nuclei of epithelial cells. A noticeable difference was observed between the groups, as in calves, unlike in adults, the parietal cells were stained, and the reaction was generally denser than that in adult individuals (Fig. 1 D-E). These cells exhibited a strong presence of PNX throughout the mucosal layer. GPR173 receptor localization in the abomasum was present in glandular epithelial cells, affecting the cytoplasm and single nuclei (Fig. 2D 1 ). The receptor was found as either diffuse or focal granular structures. Additionally, blood vessels in the abomasum showed GPR173 localization throughout their walls, including the endothelium, although the intensity and coverage varied among different vessels, and not all vessels were found to have IR (Fig. 2D 2 ). Small intestine: duodenum, jejunum, and ileum In the small intestine, PNX-IR and GPR173 receptor localization were consistent across the duodenum, jejunum, and ileum. PNX-positive cells were infrequent and often appeared as single cells in the glandular epithelium, primarily in the enteroendocrine cells (Fig. 3 A-C). In the duodenum, jejunum and ileum, IR localization was similar, with a predominantly cytoplasmic distribution, whereas in the duodenum, IR was also observed in the duodenal glands. For GPR173, the receptor was intensely localized in the mucosal epithelium and lamina propria (Fig. 4A 1 , B 1 , C 1 ). In the duodenum, the cells exhibited strong granular localization in the cytoplasm and nucleus. The jejunum and ileum exhibited similar patterns, with the receptor present in both the cytoplasm and nuclei of glandular epithelial cells and lamina propria cells in both age groups. The blood vessels across these sections showed GPR173 localization throughout their walls, including the endothelium, although IR was not found in every vessel (Fig. 4A 2 , B 2 , C 2 ). Large intestine: colon In the colon, PNX and GPR173 localization mirrored that observed in the small intestine. PNX-positive cells were located in the glandular epithelium and showed a sparse cytoplasmic distribution similar to that in the small intestine (Fig. 3 D). In this section, PNX-IR cells were also noted in the lamina propria between the glands. GPR173 receptor localization in the colonic mucosa was evident in the cytoplasm and nuclei of glandular epithelial cells, as well as in lamina propria cells, which was consistent with the patterns observed in the jejunum and ileum (Fig. 4D 1 ). The blood vessels in the colon exhibited strong receptor localization throughout their walls, including the endothelium, similar to other intestinal sections (Fig. 4D 2 ). Neuronal reactions Throughout the GIT, GPR173 displayed prominent localization in the nerve fibres within both the submucosal and muscular plexuses (Fig. 2A 3 , B 3 , C 3 , D 3 ; Fig. 4A 3 , B 3 , C 3 , D 3 ). These reactions were intense and uneven, affecting fibres encircling nerve ganglia and fibres within the muscular, submucosal, and mucosal layers. In the intestines, single neurons in the small intestine and multiple neurons in the colon exhibited receptor localization. Enzyme-linked immunosorbent assay (ELISA) PNX In calves, the reticulum and abomasum presented the highest concentration of PNX-14, with slightly lower, but not statistically significant, levels in the rumen (Fig. 5 ). Significantly lower concentrations were detected in the omasum, duodenum, jejunum, ileum, and colon relative to the reticulum and abomasum, with the ileum and colon showing the lowest levels of PNX. Significant differences were identified between the following segments: rumen and omasum (P = 0.007), rumen and ileum (P = 0.003), rumen and colon (P < 0.001), reticulum and omasum (P < 0.001), reticulum and duodenum (P = 0.043), reticulum and jejunum (P = 0.014), reticulum and ileum (P < 0.001), reticulum and colon (P < 0.001), omasum and abomasum (P < 0.001), omasum and duodenum (P = 0.024), abomasum and duodenum (P = 0.047), abomasum and jejunum (P = 0.021), abomasum and ileum (P < 0.001), abomasum and colon (P < 0.001), duodenum and ileum (P = 0.011), duodenum and colon (P = 0.003), jejunum and ileum (P = 0.044), and jejunum and colon (P = 0.008). In adult cattle, PNX-14 concentrations were highest in the reticulum, followed by the rumen and omasum (Fig. 5 ). The remaining GIT segments presented similar yet low PNX-14 concentrations. Statistically significant differences were detected between the rumen and other segments: rumen vs. reticulum (P = 0.05) and P < 0.001 for comparisons with the remaining segments, such as the reticulum and other segments (P < 0.001) and the omasum and other segments (P < 0.001). Comparative analysis between calves and adults revealed significantly higher PNX-14 levels in adults within the rumen, reticulum and omasum (P < 0.001) but in calves within the abomasum, duodenum, jejunum, and colon (P = 0.005 for the colon and P < 0.001 for the abomasum, duodenum, and jejunum) (Fig. 5 ). GPR173 In calves, the rumen presented the highest concentration of GPR173, with slightly lower, yet not statistically significant, levels observed in the reticulum, omasum, and abomasum (Fig. 6 ). The lowest concentrations were found in the intestines. Significant differences in GPR173 levels were identified between the following segments: the rumen and duodenum (P = 0.002), the rumen and jejunum (P < 0.001), the rumen and ileum (P = 0.016), the rumen and colon (P = 0.010), and the omasum and jejunum (P = 0.023). In adult cattle, the omasum presented the highest concentration of GPR173, followed by the reticulum, abomasum, and duodenum, with no significant differences among them (Fig. 6 ). The remaining segments of the GIT presented the lowest levels of GPR173. Significant differences were noted between the following segments: the rumen and omasum (P = 0.004), the reticulum and jejunum (P = 0.005), the reticulum and ileum (P = 0.007), the omasum and jejunum (P < 0.001), the omasum and ileum (P < 0.001), and the omasum and colon (P = 0.005). Comparative analysis between age groups revealed a significant difference in GPR173 levels in the duodenum, with adults exhibiting higher levels than calves (P = 0.018) (Fig. 6 ). Discussion The findings from this study provide a comprehensive analysis of the immunolocalization and immunoexpression levels of PNX-14 and its receptor GPR173 across various segments of the bovine GIT in both calves and adult cattle. The differential expression patterns observed suggest potentially significant functional roles for these molecules in bovine GIT physiology and possibly reveal age-related differences in their regulatory mechanisms. Additionally, the methodologies employed in this study, including immunohistochemistry (IHC) and ELISA, provide a framework for confirming the localization and immunoexpression levels of PNX-14 and GPR173, lending credibility to the conclusions drawn ( 5 , 12 ). The levels of PNX-14 were higher in calves compared to adults in all segments except the forestomachs, where adults presented greater concentrations. This finding provides valuable insights into the peptide's potential roles, as the rumen, reticulum, and omasum have limited activity in calves and gradually develop their functions with the introduction of solid feed ( 13 ). This pattern suggests that PNX-14 may play a significant role in food processing. In support of this hypothesis, PNX has also been detected in premetazoans, where it is believed to influence food intake ( 1 ). However, this does not preclude the function of the peptide in the forestomachs of calves, as its levels in the rumen and reticulum were still higher than in the intestines. These findings indicate that PNX-14 may contribute to the early development of these compartments. The eight-month-old calves in this study had transitioned from monogastric to polygastric digestion, suggesting that their forestomachs were functional but not yet fully mature compared with those of adults. This transition is reflected in the higher levels of PNX-14 observed in the rumen, reticulum, and omasum of adult cattle. These compartments play crucial roles in microbial fermentation and nutrient absorption, processes that require mucosal protection and regulation. The increased PNX-14 levels in these compartments in adults likely support these functions, helping to maintain mucosal integrity and manage the inflammatory responses associated with microbial activity. The protective effects of PNX-14 against gastrointestinal stressors highlighted by Zandeh-Rahimi et al. in 2022 ( 8 ) demonstrated its ability to significantly reduce macroscopic duodenal lesions, lower the serum levels of proinflammatory cytokines, and decrease the activity of oxidative stress markers such as malondialdehyde and myeloperoxidase in rats. However, the lower levels of PNX-14 detected in the bovine GIT compared with the higher concentrations administered extrinsically in Zandeh-Rahimi et al.'s study suggest that these protective benefits might not be as pronounced in cattle. The physiological conditions in bovines likely involve much lower concentrations of PNX-14, which may not be sufficient to elicit the same extent of protective effects observed in the rat model. This disparity indicates that while PNX-14 could offer some benefits in mitigating gastrointestinal stressors, its impact on cattle may be limited due to its lower endogenous levels. Consequently, the protective role of PNX-14 in the bovine GIT may be less significant, necessitating further research to explore the specific conditions under which PNX-14 might exert its beneficial effects. The higher PNX-14 levels in the abomasum of calves suggest that PNX-14 may play a role in protecting the mucosal lining and modulating inflammation in this critical digestive compartment as calves transition from milk to solid feed ( 14 ). The difference in the level of PNX was also noticeable in the IHC study. The abomasum of calves was characterized by the presence of PNX-IR within parietal cells. It can therefore be speculated that in young cattle, PNX somehow aids in regulating gastric acid secretion and, consequently, supports digestive and antimicrobial protective functions ( 15 ). There are no studies available on the concentration of PNX in the GIT in cattle. However, a study conducted in rats by discoverers of PNX demonstrated the highest levels in the stomach, followed by the jejunum, duodenum, ileum, and colon ( 2 ). Furthermore, the protein levels obtained in the present study are consistent with those reported in a previous study assessing the mRNA levels of the precursor SMIM20, as SMIM20 mRNA was most abundant in the rumen and reticulum ( 12 ). Notably, the difference between mRNA and protein levels is highlighted by the lack of statistically significant differences in mRNA levels between adult individuals and calves ( 12 ). Immunolocalization of PNX-14 observed exclusively in the mucosa of all sections of the GIT in relatively small amounts (approximately 1 IR cell per 100 cells in most cases) may also support the notion that it does not play significant roles in the cattle GIT. On the other hand, while the levels of PNX-14 detected in the bovine GIT are relatively low, this does not necessarily imply that its role is insignificant. The small amounts of PNX-14 could be due to the small size of the peptide, but the possibility that even low concentrations can exert significant biological effects is probable. For example, interleukin-6, a protein known for its crucial role in inflammation and immune responses, also exists at low concentrations but has profound impacts on physiological processes ( 16 ). This, in turn, may support the possibility that even small amounts of PNX-14 could play important roles in maintaining gut health and regulating various functions within the GIT. Another possible explanation could be the release of PNX under specific conditions (as referenced in previous studies, such as during stress or inflammation), which were not met in this study because the animals were healthy ( 8 , 17 ). Furthermore, since PNX is an orexigenic peptide that inhibits appetite ( 18 ), it is possible that its overproduction occurs postprandially, which we could not assess owing to the fasting of the animals before slaughter. An immunolocalization study of PNX-14 in the rat GIT demonstrated the presence of PNX-14 between the intestinal glands and the lamina propria of the mucosa but not in epithelial cells. Interestingly, no IR was detected in the stomach or colon ( 4 ). Our study differs from the aforementioned research, as we observed an IR in all segments of the GIT, from the stomach to the colon. Additionally, we noted PNX-IR in the intestinal glands, as well as an IR between the glands in the colon. This particular localization in the lamina propria suggests that PNX-14 may be potentially involved in immune functions, as it is located in areas populated by cells usually responsible for the immune response ( 19 ). Given the observed discrepancies in the research, further exploration of this topic is clearly needed. Conducting additional analyses involving other species will be essential to deepen our understanding and address the existing gaps in knowledge. GPR173 showed distinct localization patterns throughout the entire GIT, particularly in the nerve fibres within both the submucosal and muscular plexuses. The prominent localization of GPR173 in neural tissues indicates that it plays a significant role in modulating gut motility and neural signalling. The uneven reactions of GPR173 in nerve fibres suggest that its activity might be finely tuned to specific physiological conditions, contributing to the adaptive responses of the GIT to various stimuli. Additionally, the reaction was incidentally present in individual neurons of the intestines, indicating nonuniform localization of the receptor. This finding aligns with the established roles of GPCRs in the enteric nervous system, where they modulate various aspects of gut motility and secretion ( 20 ). Additionally, GPR173 was observed in epithelial cells and blood vessels throughout the entire GIT, suggesting its involvement in epithelial and vascular functions. In all segments of the intestine, IR was also noted in the lamina propria, possibly in immune cells. The granular presence of the receptor in these areas, coupled with the low levels of PNX-14, implies that GPR173 might interact with other ligands to fulfil its roles and the fact that GPR173 levels were much higher than PNX-14 levels (PNX-14 in pg/mg of total protein, whereas GPR173 in ng/mg of total protein) further support this notion. Notably, He et al. postulated that GPR173 is a receptor for CCK, which is known to be an important gastrointestinal hormone ( 11 ). This could be a plausible explanation for the widespread distribution of GPR173 observed in our study. The significant differences in GPR173 levels between segments such as the rumen and intestines highlight the receptor’s segment-specific roles, suggesting broader mechanisms of nutrient regulation and absorption in the bovine GIT. The age-related differences in GPR173 levels, particularly the higher levels in the duodenum of adults than in those of calves, suggest developmental changes in digestive physiology and nutrient absorption efficiency. This differential expression indicates a potential adaptation to increased metabolic demands in adults. The localization of GPR173 aligns with the observations of Aleti et al., indicating that GPCR family receptors might mediate interactions between microbial metabolites and human cells and could be responsible for regulating the gut–immune–brain axis ( 21 ). There are no other studies describing the immunolocalization of GPR173 in the GIT. However, our study on the expression of GPR173 mRNA in the bovine GIT revealed somewhat different expression patterns compared with the protein concentration. For both mRNA and protein, the highest levels were noted in the omasum. However, the mRNA levels were much more variable between different sections compared to the protein levels, which were relatively consistent across various sections of the GIT ( 12 ). Our observation that PNX-14 and GPR173 are consistently present throughout the GIT supports their peripheral roles, possibly distinct from their central effects on food intake regulation. These findings align with those of Schalla et al. ( 18 ), who reported that PNX-14 does not cross the blood‒brain barrier in vitro, potentially indicating its localized action in peripheral tissues. This, in turn, may support the notion that PNX-14 might not function as a gut–brain peptide, with its effects being confined to peripheral tissues rather than influencing central nervous system processes. Moreover, the documented challenges of intraperitoneal administration to induce the expected orexigenic effect of PNX ( 5 , 22 ) and the peptide’s ability to stimulate insulin secretion ( 3 ) suggest that PNX’s functional scope extends to metabolic regulation within the GIT. Intraperitoneal administration difficulties may arise from the challenges in ensuring effective delivery and bioavailability of PNX-14, possibly due to its rapid degradation or insufficient targeting of specific receptor sites within the GIT. This suggests that PNX-14’s effects are highly context-dependent and may require precise delivery mechanisms to elicit significant biological responses. The peptide's ability to stimulate insulin secretion also points to a broader role in metabolic regulation, potentially influencing glucose homeostasis and energy balance within the GIT. This metabolic role may involve intricate interactions with other gut hormones and regulatory peptides, further underscoring the complexity of PNX-14’s functions beyond its primary sites of action. One of the limitations of this study is that PNX-14 is the shortest form of PNX, which may cause an antibody directed against PNX-14 to also bind to the longer forms of PNX. Another previously mentioned limitation is that PNX has orexigenic properties and may be released postprandially. However, only fasted animals could be used in the study. Conclusion In conclusion, despite the low concentration of PNX-14 in the bovine GIT, it cannot be ruled out that it may play a significant role in digestive processes or the immune response. Conversely, GPR173 is more abundant, indicating that it may interact with other ligands and contribute to GIT functions through broader mechanisms. The peripheral roles of PNX-14 and GPR173, which are distinct from their central nervous system effects, suggest localized actions within the GIT that merit further investigation. The complexities of their interactions and the context-dependent nature of their effects highlight the need for precise delivery mechanisms and a deeper understanding of their roles in bovine health and nutrition. This study fills a gap in the information concerning the localization and levels of PNX-14 and its receptor GPR173 in the GIT of domestic cattle. On the basis of these results, further research can undoubtedly be conducted in other species, as well as in domestic cattle. The high levels of PNX in forestomachs, which are unique to ruminants, may indicate that PNX has important functions in polygastric animals. Methods Animals and tissue samples This study involved healthy male Polish Holstein–Friesian cattle, including two distinct age groups: six adult animals aged 20–24 months, weighing 768 ± 46 kg, and six calves aged 7–8 months, weighing 218 ± 23 kg. All the subjects were sourced from a single farm, ensuring uniform living conditions, diet, and environmental factors. Both groups followed a semi-intensive feeding regimen, consisting of initial grazing on pasture followed by the total mixed ration feeding method, as described by Włodarczyk et al. in 2011 ( 23 ). Tissue samples were collected at a local slaughterhouse. Prior to slaughter, the cattle were fasted for 18 hours. Next, tissue sections were obtained from various segments of the GIT, including the rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, and colon. Care was taken to include all layers of each GIT section in the excised samples. Immediately after collection, the tissues were rinsed with a physiological saline solution to remove any residual contents and contaminants. Part of the material designated for ELISA testing was immediately placed in liquid nitrogen and then transferred to -80°C, while samples intended for IHC studies were placed in buffered 4% formaldehyde. Additionally, postmortem examinations confirmed the good health of the animals and revealed no pathologies within their digestive tracts, confirming the validity of the collected samples for this study. Given that tissue collection was conducted postmortem, ethical review and approval by the Ethics Committee were not required under Polish law. Tissue processing Samples fixed for 24 h in formaldehyde were rinsed in tap water, dehydrated in an increasing ethanol series, cleared in xylene and infiltrated with paraffin. The fixed samples were then embedded in paraffin via a modular embedding center (MYR EC-350, Casa Álvarez Material Científico SA, Madrid, Spain) and subsequently sliced into 5 µm-thick sections via a rotary microtome (HM 360, Microm, Walldorf, Germany). Every fifth slice was placed on SuperFrost® Plus slides (Thermo Scientific, Menzel-Glaser, Braunschweig, Germany) and stored in an incubator (CG Wamed, Warsaw, Poland) at 37°C for 12 h. From each frozen sample, 100 mg was weighed and homogenized in phosphate-buffered saline (PBS, 0.1 M, pH = 7.3) at a ratio of 1:9 (tissue:PBS). The homogenates were centrifuged at 12,000 × g for 10 minutes at 4°C to remove debris, and the supernatants were collected. The protein content was subsequently determined using Pierce BCA kit (Thermo Scientific, Waltham, MA, USA) and expressed in µg/ml. IHC For this study, IHC analysis was carried out to detect PNX-14 and GPR173 in the GIT tissues of calves and adult domestic cattle. The IHC analysis followed the protocol described by Osiak-Wicha et al., ( 24 ) with additional steps utilizing xylene for dewaxing the slides and rehydration in a descending concentration of ethyl alcohol. During the first day, using the sodium-citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH = 6.0), heat-induced epitope retrieval was performed (8 min at 80°C, multicooker RMC-PM381-E Redmond, China), followed by a 10-minute incubation in 3% H 2 O 2 . Next, after blocking in UltraVision Protein Block (5 min; Thermo Scientific, Waltham, MA, USA), an overnight incubation in 4°C with primary antibodies directed against PNX-14 and GPR173 was carried out (Table 1 ). Only one primary antibody per section was used. The next day, the sections were incubated with a BrightVision two-step detection system of poly-HRP-anti-Ms/Rb IgG (ImmunoLogic WellMed B.V., Duiven, Netherlands; Table 1 ) for a total of 45 min, and immunostaining was performed with 3,3′-diaminobenzidine (DAB substrate kit; ab64238; Abcam, Cambridge, UK). PBS was used to rinse the slides between each step. After DAB staining, the sections were washed with distilled water, counterstained with Meyer’s hematoxylin (Patho, Mar-Four, Konstantynów Łódzki, Poland), washed with tap water for 10 minutes, dehydrated with ascending concentrations of ethyl alcohol, cleared with xylene, cover-slipped with Shandon Consul-Mount (Thermo Scientific, Waltham, MA, USA), and dried in an incubator (CG Wamed, Warsaw, Poland) at 37°C for 12 h. The specificity of the antibodies was evaluated through two methods: a negative control, where the primary antibodies were substituted with the antibody diluent, and a preabsorption experiment, where the primary antibodies were mixed with an excess of the target synthetic protein before incubation. No positive IR was observed in any of the control sections. Table 1 Primary and secondary antibodies used in the study. Antibody Host Catalog number Dilution Manufacturer Primary antibody Anti – phoenixin-14 amide rabbit H-079-01 1:200 Phoenix Pharmaceuticals, Burlingame, CA, US Anti-GPR173 rabbit PA5-33664 8.4 µg/mL Thermo Scientific, Menzel-Glaser, Braunschweig, Germany Secondary antibody Anti-mouse/Anti-rabbit goat DPVB-HRP RTU 1 ImmunoLogic, Duiven, Netherlands 1 RTU = Ready To Use The stained slides were examined under a light microscope (BX-51 DSU, Olympus, Tokyo, Japan) equipped with a digital color camera (DP-70, Olympus, Tokyo, Japan). High-resolution digital photographs were taken using Cell^M 2.3 software (Olympus cellSens Standard) under consistent lighting conditions and uniform settings for brightness and contrast by a single individual. ELISA PNX-14 and GPR173 levels were determined in duplicate using commercially available kits (EH4521, FineTest, Wuhan, China – PNX-14; MBS9330587, MyBioSource, San Diego, CA, USA – GPR173) according to the manufacturer's protocol. The absorbance values were measured using the Epoch Microplate Spectrophotometer (BioTek, Winooski, VT, USA). The intra-assay coefficient of variation did not exceed 8% in either measurement, and the sensitivity of the kits was 0.938 pg/ml for PNX-14 and 0.1 ng/ml for GPR173. Statistical analysis The concentration of the target analyte in each sample was normalized to the total protein content. The results are expressed as pg or ng of target analyte per mg of total protein (pg/mg protein for PNX-14 and ng/mg protein for GPR173). The average results for each sample were analysed using two-way analysis of variance (ANOVA), followed by Tukey’s HSD post hoc test. Homogeneity of variance was tested using Levene's test. A p-value < 0.05 was considered statistically significant. All the statistical analyses were conducted using GraphPad Prism, version 9.5.0 for Windows (GraphPad Software, San Diego, CA, US; www.graphpad.com ; accessed on June 10, 2024). Abbreviations PNX phoenixin SMIM20 small integral membrane protein 20 GIT gastrointestinal tract ELISA enzyme–linked immunosorbent assay IHC immunohistochemistry HPG hypothalamic‒pituitary‒gonadal SREB3 super–conserved receptor expressed in the brain 3 GPCR G protein–coupled receptors CCK cholecystokinin IR immunoreactivity PBS phosphate–buffered saline Declarations Ethics approval and consent to participate According to Polish law, since all tissue collection procedures were conducted post-mortem, ethical review and approval from the Ethics Committee for this study were not required. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article. Competing interests The authors declare that they have no competing interests. Funding This research was supported by project no. WKN/MN-1/WET/22, provided by the University of Life Sciences in Lublin, Poland, for Katarzyna Kras. Authors' contributions K.K.: conceptualization, study design, methodology, writing—original draft preparation, data analysis, visualization; C.O.-W.: statistical analysis of data, writing—reviewing and editing; M.B.A.: conceptualization, writing—reviewing and editing, supervision. All authors have read and approved the final manuscript. Acknowledgements The authors would like to express sincere gratitude to Professor Ewa Tomaszewska for her invaluable substantive support throughout the course of this research. References Yañez-Guerra LA, Thiel D, Jékely G. Premetazoan Origin of Neuropeptide Signaling. Mol Biol Evol. 2022;39(4):msac051. Yosten GLC, Lyu RM, Hsueh AJW, Avsian-Kretchmer O, Chang JK, Tullock CW, et al. A novel reproductive peptide, phoenixin. J Neuroendocrinol. 2013;25(2):206–15. Billert M, Rak A, Nowak KW, Skrzypski M. Phoenixin: More than Reproductive Peptide. Int J Mol Sci. 2020;21(21):8378. Prinz P, Scharner S, Friedrich T, Schalla M, Goebel-Stengel M, Rose M, et al. Central and peripheral expression sites of phoenixin-14 immunoreactivity in rats. Biochem Biophys Res Commun. 2017;493(1):195–201. Schalla MA, Stengel A. Phoenixin-A Pleiotropic Gut-Brain Peptide. Int J Mol Sci. 2018;19(6):1726. Liang H, Zhao Q, Lv S, Ji X. Regulation and physiological functions of phoenixin. Front Mol Biosci. 2022;9:956500. Mcilwraith EK, Belsham DD. Phoenixin: uncovering its receptor, signaling and functions. Acta Pharmacol Sin. 2018;39(5):774–8. Zandeh-Rahimi Y, Panahi N, Hesaraki S, Shirazi-Beheshtiha SH. Protective Effects of Phoenixin-14 Peptide in the Indomethacin-Induced Duodenal Ulcer: An Experimental Study. Int J Pept Res Ther. 2022;28(1):43. Kupcova I, Danisovic L, Grgac I, Harsanyi S. Anxiety and Depression: What Do We Know of Neuropeptides? Behav Sci Basel Switz. 2022;12(8):262. McIlwraith EK, Zhang N, Belsham DD. The Regulation of Phoenixin: A Fascinating Multidimensional Peptide. J Endocr Soc. 2022;6(2):bvab192. He L, Shi H, Zhang G, Peng Y, Ghosh A, Zhang M, et al. A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition. J Neurosci Off J Soc Neurosci. 2023;43(13):2305–25. Kras K, Ropka-Molik K, Muszyński S, Arciszewski MB. Expression of Genes Encoding Selected Orexigenic and Anorexigenic Peptides and Their Receptors in the Organs of the Gastrointestinal Tract of Calves and Adult Domestic Cattle ( Bos taurus taurus ). Int J Mol Sci. 2023;25(1):533. Govil K, Yadav D, Patil A, Nayak S, Baghel R, Yadav P, et al. Feeding management for early rumen development in calves. J Entomol Zool Stud. 2017;5(3):1132–9. Pisoni L, Relling AE. The effects of supplementing yeast fermentation products on gut permeability, hormone concentration, and growth in newborn dairy calves. Transl Anim Sci. 2020;4(2):809–21. Engevik AC, Kaji I, Goldenring JR. The Physiology of the Gastric Parietal Cell. Physiol Rev. 2020;100(2):573–602. Tomaszewska E, Świątkiewicz M, Muszyński S, Donaldson J, Ropka-Molik K, Arciszewski MB, et al. Repetitive Cerulein-Induced Chronic Pancreatitis in Growing Pigs-A Pilot Study. Int J Mol Sci. 2023;24(9):7715. Friedrich T, Stengel A. Current state of phoenixin-the implications of the pleiotropic peptide in stress and its potential as a therapeutic target. Front Pharmacol. 2023;14:1076800. Schalla MA, Oerter S, Cubukova A, Metzger M, Appelt-Menzel A, Stengel A. Locked Out: Phoenixin-14 Does Not Cross a Stem-Cell-Derived Blood–Brain Barrier Model. Brain Sci. 2023;13(7):980. Montalban-Arques A, Chaparro M, Gisbert JP, Bernardo D. The Innate Immune System in the Gastrointestinal Tract: Role of Intraepithelial Lymphocytes and Lamina Propria Innate Lymphoid Cells in Intestinal Inflammation. Inflamm Bowel Dis. 2018;24(8):1649–59. Weinberg ZY, Puthenveedu MA. Regulation of G protein-coupled receptor signaling by plasma membrane organization and endocytosis. Traffic Cph Den. 2019;20(2):121–9. Aleti G, Troyer EA, Hong S. G protein-coupled receptors: A target for microbial metabolites and a mechanistic link to microbiome-immune-brain interactions. Brain Behav Immun - Health. 2023;32:100671. Schalla M, Prinz P, Friedrich T, Scharner S, Kobelt P, Goebel-Stengel M, et al. Phoenixin-14 injected intracerebroventricularly but not intraperitoneally stimulates food intake in rats. Peptides. 2017;96:53–60. Włodarczyk R, Budvytis M. Proper nutrition for high yielding cows - how to fully utilize their production potential. Vet Life. 2011;86:771–6. Osiak-Wicha C, Muszyński S, Tomaszewska E, Kras K, Ropka-Molik K, Zhyla M, et al. Gene Expression Level and Immunohistochemical Localization of Cannabinoid and Cannabinoid-Related Receptors in The Small Intestine of Holstein Bulls ( Bos taurus taurus ). Ann Anim Sci. 2024;24(3):779–89. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 18 Feb, 2025 Read the published version in BMC Veterinary Research → Version 1 posted Editorial decision: Revision requested 30 Sep, 2024 Reviewers agreed at journal 25 Sep, 2024 Reviews received at journal 25 Sep, 2024 Reviewers agreed at journal 18 Sep, 2024 Reviewers agreed at journal 03 Sep, 2024 Reviews received at journal 20 Aug, 2024 Reviewers agreed at journal 11 Aug, 2024 Reviewers agreed at journal 11 Aug, 2024 Reviewers invited by journal 08 Aug, 2024 Editor invited by journal 08 Aug, 2024 Editor assigned by journal 06 Aug, 2024 Submission checks completed at journal 06 Aug, 2024 First submitted to journal 03 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4852060","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":347968316,"identity":"f918f04a-0f33-4134-8035-4c9d8ebb3f9c","order_by":0,"name":"Katarzyna Kras","email":"data:image/png;base64,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","orcid":"","institution":"University of Life Sciences in Lublin","correspondingAuthor":true,"prefix":"","firstName":"Katarzyna","middleName":"","lastName":"Kras","suffix":""},{"id":347968317,"identity":"caa5f50f-24d7-403d-a9e2-3940af751466","order_by":1,"name":"Cezary Osiak-Wicha","email":"","orcid":"","institution":"University of Life Sciences in Lublin","correspondingAuthor":false,"prefix":"","firstName":"Cezary","middleName":"","lastName":"Osiak-Wicha","suffix":""},{"id":347968318,"identity":"d01fcb68-8322-46fb-a82f-8a11505e87c0","order_by":2,"name":"Marcin B. Arciszewski","email":"","orcid":"","institution":"University of Life Sciences in Lublin","correspondingAuthor":false,"prefix":"","firstName":"Marcin","middleName":"B.","lastName":"Arciszewski","suffix":""}],"badges":[],"createdAt":"2024-08-03 07:45:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4852060/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4852060/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-025-04545-x","type":"published","date":"2025-02-18T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63831485,"identity":"7d2a7e03-d9fa-4c43-ace9-6bef42f7ed63","added_by":"auto","created_at":"2024-09-02 19:08:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3114109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunolocalization of phoenixin-14 (PNX-14) in the stomach of domestic cattle. \u003c/strong\u003eImmunoreactions in the rumen (A), reticulum (B), omasum (C) and abomasum (D and E) in calves (A, B and D) and adult (C and E) domestic cattle. Black arrows indicate immunoreactions in the epithelial cells, red arrows indicate parietal cells. Scale bar: 40 µm.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4852060/v1/d58d8597dbdc4096d6e1ba75.jpg"},{"id":63831488,"identity":"ccf01838-67de-4c45-91f7-f043a40cec2b","added_by":"auto","created_at":"2024-09-02 19:08:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4189543,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunolocalization of GPR173 in the stomach of domestic cattle. \u003c/strong\u003eImmunoreactions in the rumen (A\u003csub\u003e1-3\u003c/sub\u003e), reticulum (B\u003csub\u003e1-3\u003c/sub\u003e), omasum (C\u003csub\u003e1-3\u003c/sub\u003e) and abomasum (D\u003csub\u003e1-3\u003c/sub\u003e) in calves (A\u003csub\u003e2\u003c/sub\u003e, A\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e, D\u003csub\u003e1\u003c/sub\u003e, D\u003csub\u003e2\u003c/sub\u003e, D\u003csub\u003e3\u003c/sub\u003e) and adult (A\u003csub\u003e1\u003c/sub\u003e, B\u003csub\u003e1\u003c/sub\u003e, B\u003csub\u003e2\u003c/sub\u003e, B\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e1\u003c/sub\u003e) domestic cattle. Black arrows indicate immunoreactions in the epithelial cells (A\u003csub\u003e1\u003c/sub\u003e, B\u003csub\u003e1\u003c/sub\u003e, C\u003csub\u003e1\u003c/sub\u003e, D\u003csub\u003e1\u003c/sub\u003e), blood vessels (A\u003csub\u003e2\u003c/sub\u003e, B\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e2\u003c/sub\u003e, D\u003csub\u003e2\u003c/sub\u003e), and nerve fibers (A\u003csub\u003e3\u003c/sub\u003e, B\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e, D\u003csub\u003e3\u003c/sub\u003e). Scale bar: 40 µm.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4852060/v1/fe374da9a798d08edfafab39.jpg"},{"id":63831489,"identity":"a7771458-a9b0-4976-b903-83c73208c191","added_by":"auto","created_at":"2024-09-02 19:08:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2840610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunolocalization of phoenixin-14 (PNX-14) in the intestines of domestic cattle. \u003c/strong\u003eImmunoreactions in the duodenum (A), jejunum (B), ileum (C) and colon (D) in calves (A, B, D) and adult (C) domestic cattle. Black arrows indicate immunoreactions in the epithelial cells (A, B, and C), and lamina propria cell (D). Scale bar: 40 µm.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4852060/v1/3c750b2d0fbd8bd247599ca4.jpg"},{"id":63832381,"identity":"05f26a93-9caa-4e74-ae9c-2cfac9b72bbb","added_by":"auto","created_at":"2024-09-02 19:16:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3717109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunolocalization of GPR173 in the intestines of domestic cattle.\u003c/strong\u003e Immunolocalization in the duodenum (A\u003csub\u003e1-3\u003c/sub\u003e), jejunum (B\u003csub\u003e1-3\u003c/sub\u003e), ileum (C\u003csub\u003e1-3\u003c/sub\u003e) and colon (D\u003csub\u003e1-3\u003c/sub\u003e) in calves (A\u003csub\u003e2\u003c/sub\u003e, A\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e, D\u003csub\u003e1\u003c/sub\u003e, D\u003csub\u003e2\u003c/sub\u003e, D\u003csub\u003e3\u003c/sub\u003e) and adult (A\u003csub\u003e1\u003c/sub\u003e, B\u003csub\u003e1\u003c/sub\u003e, B\u003csub\u003e2\u003c/sub\u003e, B\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e1\u003c/sub\u003e) domestic cattle. Black arrows indicate immunoreactions in the epithelial cells (A\u003csub\u003e1\u003c/sub\u003e, B\u003csub\u003e1\u003c/sub\u003e, C\u003csub\u003e1\u003c/sub\u003e, D\u003csub\u003e1\u003c/sub\u003e), blood vessels (A\u003csub\u003e2\u003c/sub\u003e, B\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e2\u003c/sub\u003e, D\u003csub\u003e2\u003c/sub\u003e), and nerve fibers (A\u003csub\u003e3\u003c/sub\u003e, B\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e, D\u003csub\u003e3\u003c/sub\u003e). Scale bar: 40 µm.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4852060/v1/233a74ad96c1adc836815df8.jpg"},{"id":63832380,"identity":"a0d8dd9d-7528-4602-9253-48883f474f74","added_by":"auto","created_at":"2024-09-02 19:16:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":130922,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhoenixin-14 concentration [pg/mg total protein] in the gastrointestinal tract (GIT) of calves and adult cattle\u003c/strong\u003e. Different lowercase letters denote significant differences between GIT segments in calves, whereas different uppercase letters denote significant differences between GIT segments in adults (p \u0026lt; 0.05). The asterisks (*) highlight significant differences in the phoenixin-14 concentration between calves and adults within a specific GIT segment (** for p \u0026lt; 0.01; *** for p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4852060/v1/34971ace80a5d839a702a42a.jpg"},{"id":63831483,"identity":"8d83effb-6c82-497c-bf6a-195243e53a43","added_by":"auto","created_at":"2024-09-02 19:08:55","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":121466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPR173 concentration [ng/mg total protein] in the gastrointestinal tract (GIT) of calves and adult cattle\u003c/strong\u003e. Different lowercase letters denote significant differences between GIT segments in calves, whereas different uppercase letters denote significant differences between GIT segments in adults (p \u0026lt; 0.05). The asterisk (*) highlights significant differences in the GPR173 concentration between calves and adults within a specific GIT segment (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4852060/v1/b72df070bd862724ac956066.jpg"},{"id":77052544,"identity":"192ec27e-377b-4722-9f8a-22c1e7437738","added_by":"auto","created_at":"2025-02-24 16:14:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15017198,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4852060/v1/e837ee08-ded8-4fd8-8166-b06da0197c2c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Immunolocalization and immunoexpression levels of the novel peptide phoenixin-14 and its receptor GPR173 in the gastrointestinal tract of calves and adult domestic cattle (Bos taurus taurus)","fulltext":[{"header":"Background","content":"\u003cp\u003ePhoenixin (PNX), although probably one of the oldest discovered neuropeptides, has just recently been identified and has sparked significant interest because of its wide-ranging physiological roles and highly conserved structure across species (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). PNX is derived from the enzymatic cleavage of small integral membrane protein 20 (SMIM20), a precursor protein encoded by the \u003cem\u003eSMIM20\u003c/em\u003e gene. This cleavage results in the production of two primary isoforms: PNX-14, which is 14 amino acids long, and PNX-20, which consists of 20 amino acids (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). \u003cem\u003eSMIM20\u003c/em\u003e mRNA expression has been extensively investigated in both central and peripheral tissues, including the gastrointestinal tract (GIT), heart, and lungs. Notably, the highest expression levels were found in the hypothalamus, followed by the heart, with lower levels detected in other tissues (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The detection of PNX in various species, such as humans, rodents, pigs, cattle, and chickens, coupled with its highly conserved structure, particularly in mammals, suggests a crucial biological role for this peptide (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDiscovered in 2013 by Yosten et al. through a bioinformatic approach (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), PNX has been implicated in various regulatory functions within the body. This peptide is particularly notable for its involvement in critical processes such as food intake, reproductive function, and cardiovascular regulation (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). As research into PNX progresses, its potential impact on different bodily systems is becoming more evident, with emerging studies highlighting its roles in mental health, including anxiety and depression (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Nevertheless, the functional repertoire of PNX is expanding as new roles are continuously uncovered. However, the role of PNX in the GIT remains elusive due to the scarcity of related research. Preliminary findings suggest a protective role for PNX against duodenal ulcers (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Notably, the regulation of energy balance, including food intake, is intrinsically linked to reproductive functions and is mediated through the hypothalamic‒pituitary‒gonadal (HPG) axis. This axis, in turn, is also connected with anxiety and depression, highlighting the multifaceted interactions among these processes (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePNX exerts its effects primarily through the GPR173 receptor, also known as the super-conserved receptor expressed in the brain 3 (SREB3), which is part of the extensive family of G protein-coupled receptors (GPCRs). Localization studies of GPR173 have confirmed its presence in the hypothalamus, pituitary gland, and ovaries, supporting its association with the HPG axis (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Additionally, GPR173 is expressed in the heart, skin, and pancreas (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Recent findings suggest a dual role for GPR173, not only as a receptor for PNX but also as a potential receptor for cholecystokinin (CCK), although this remains a point of contention (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the detection of \u003cem\u003eSMIM20\u003c/em\u003e and \u003cem\u003eGPR173\u003c/em\u003e mRNA across all segments of the GIT in both calves and adult cattle (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), there is a notable gap in our understanding of the actual presence and immunolocalization of the PNX and GPR173 proteins in these tissues. This study aims to fill this gap by investigating the immunolocalization and immunoexpression levels of PNX-14 and GPR173 in distinct segments of the GIT of calves and adult cattle. Given the observed presence of \u003cem\u003eSMIM20\u003c/em\u003e mRNA throughout the GIT in ruminants, we hypothesize that PNX-14 and its receptor GPR173 are immunolocalized in distinct patterns across different segments of the GIT in these animals. We further propose that the levels of immunoexpression of these proteins vary between calves and adult cattle, reflecting the developmental differences in metabolic activity and gastrointestinal function. Specifically, we expect higher immunoexpression levels in segments of the GIT that are more metabolically active or structurally complex, such as the rumen in adults and the abomasum in calves. This differential expression may indicate specialized roles for PNX-14 and GPR173 in regulating the physiological processes of digestion and nutrient absorption during different stages of development. Understanding these patterns could provide valuable insights into the functional roles of PNX-14 and GPR173 in ruminant physiology and inform strategies for optimizing nutrition and health in livestock.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePNX and GPR173 immunolocalization\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eForestomach: rumen, reticulum, and omasum\u003c/h2\u003e \u003cp\u003eIn the forestomach compartments, both PNX-14 and GPR173 exhibited specific and distinct localization patterns. PNX immunoreactivity (IR) was restricted to the mucosal epithelium in all three compartments. In the rumen, reticulum, and omasum, PNX was observed in the cytoplasm of epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor GPR173, the receptor was present in the mucosal epithelium as granular structures occupying significant areas of the cytoplasm and single nuclei in all of the forestomach compartments (Fig.\u0026nbsp;2A\u003csub\u003e1\u003c/sub\u003e, B\u003csub\u003e1\u003c/sub\u003e and C\u003csub\u003e1\u003c/sub\u003e). GPR173 was also detected in the blood vessels (Fig.\u0026nbsp;2 A\u003csub\u003e2\u003c/sub\u003e, B\u003csub\u003e2\u003c/sub\u003e and C\u003csub\u003e2\u003c/sub\u003e). Here, it appeared as dispersed granules forming occasional fibre-like arrangements within the vessel walls, with variable intensities across different vessels. Notably, the rumen lacked receptor localization in endothelial cells, in contrast with the reticulum and omasum. Moreover, IR was not observed in every vessel (Fig.\u0026nbsp;2A\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAbomasum\u003c/h2\u003e \u003cp\u003eIn the abomasum, PNX-IR was primarily observed in the cytoplasm and nuclei of epithelial cells. A noticeable difference was observed between the groups, as in calves, unlike in adults, the parietal cells were stained, and the reaction was generally denser than that in adult individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). These cells exhibited a strong presence of PNX throughout the mucosal layer. GPR173 receptor localization in the abomasum was present in glandular epithelial cells, affecting the cytoplasm and single nuclei (Fig.\u0026nbsp;2D\u003csub\u003e1\u003c/sub\u003e). The receptor was found as either diffuse or focal granular structures. Additionally, blood vessels in the abomasum showed GPR173 localization throughout their walls, including the endothelium, although the intensity and coverage varied among different vessels, and not all vessels were found to have IR (Fig.\u0026nbsp;2D\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSmall intestine: duodenum, jejunum, and ileum\u003c/h2\u003e \u003cp\u003e In the small intestine, PNX-IR and GPR173 receptor localization were consistent across the duodenum, jejunum, and ileum. PNX-positive cells were infrequent and often appeared as single cells in the glandular epithelium, primarily in the enteroendocrine cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). In the duodenum, jejunum and ileum, IR localization was similar, with a predominantly cytoplasmic distribution, whereas in the duodenum, IR was also observed in the duodenal glands.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor GPR173, the receptor was intensely localized in the mucosal epithelium and lamina propria (Fig.\u0026nbsp;4A\u003csub\u003e1\u003c/sub\u003e, B\u003csub\u003e1\u003c/sub\u003e, C\u003csub\u003e1\u003c/sub\u003e). In the duodenum, the cells exhibited strong granular localization in the cytoplasm and nucleus. The jejunum and ileum exhibited similar patterns, with the receptor present in both the cytoplasm and nuclei of glandular epithelial cells and lamina propria cells in both age groups. The blood vessels across these sections showed GPR173 localization throughout their walls, including the endothelium, although IR was not found in every vessel (Fig.\u0026nbsp;4A\u003csub\u003e2\u003c/sub\u003e, B\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLarge intestine: colon\u003c/h2\u003e \u003cp\u003eIn the colon, PNX and GPR173 localization mirrored that observed in the small intestine. PNX-positive cells were located in the glandular epithelium and showed a sparse cytoplasmic distribution similar to that in the small intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In this section, PNX-IR cells were also noted in the lamina propria between the glands. GPR173 receptor localization in the colonic mucosa was evident in the cytoplasm and nuclei of glandular epithelial cells, as well as in lamina propria cells, which was consistent with the patterns observed in the jejunum and ileum (Fig.\u0026nbsp;4D\u003csub\u003e1\u003c/sub\u003e). The blood vessels in the colon exhibited strong receptor localization throughout their walls, including the endothelium, similar to other intestinal sections (Fig.\u0026nbsp;4D\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNeuronal reactions\u003c/h2\u003e \u003cp\u003eThroughout the GIT, GPR173 displayed prominent localization in the nerve fibres within both the submucosal and muscular plexuses (Fig.\u0026nbsp;2A\u003csub\u003e3\u003c/sub\u003e, B\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e, D\u003csub\u003e3\u003c/sub\u003e; Fig.\u0026nbsp;4A\u003csub\u003e3\u003c/sub\u003e, B\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e, D\u003csub\u003e3\u003c/sub\u003e). These reactions were intense and uneven, affecting fibres encircling nerve ganglia and fibres within the muscular, submucosal, and mucosal layers. In the intestines, single neurons in the small intestine and multiple neurons in the colon exhibited receptor localization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003ePNX\u003c/h2\u003e \u003cp\u003eIn calves, the reticulum and abomasum presented the highest concentration of PNX-14, with slightly lower, but not statistically significant, levels in the rumen (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Significantly lower concentrations were detected in the omasum, duodenum, jejunum, ileum, and colon relative to the reticulum and abomasum, with the ileum and colon showing the lowest levels of PNX. Significant differences were identified between the following segments: rumen and omasum (P\u0026thinsp;=\u0026thinsp;0.007), rumen and ileum (P\u0026thinsp;=\u0026thinsp;0.003), rumen and colon (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), reticulum and omasum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), reticulum and duodenum (P\u0026thinsp;=\u0026thinsp;0.043), reticulum and jejunum (P\u0026thinsp;=\u0026thinsp;0.014), reticulum and ileum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), reticulum and colon (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), omasum and abomasum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), omasum and duodenum (P\u0026thinsp;=\u0026thinsp;0.024), abomasum and duodenum (P\u0026thinsp;=\u0026thinsp;0.047), abomasum and jejunum (P\u0026thinsp;=\u0026thinsp;0.021), abomasum and ileum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), abomasum and colon (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), duodenum and ileum (P\u0026thinsp;=\u0026thinsp;0.011), duodenum and colon (P\u0026thinsp;=\u0026thinsp;0.003), jejunum and ileum (P\u0026thinsp;=\u0026thinsp;0.044), and jejunum and colon (P\u0026thinsp;=\u0026thinsp;0.008).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn adult cattle, PNX-14 concentrations were highest in the reticulum, followed by the rumen and omasum (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The remaining GIT segments presented similar yet low PNX-14 concentrations. Statistically significant differences were detected between the rumen and other segments: rumen vs. reticulum (P\u0026thinsp;=\u0026thinsp;0.05) and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for comparisons with the remaining segments, such as the reticulum and other segments (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and the omasum and other segments (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eComparative analysis between calves and adults revealed significantly higher PNX-14 levels in adults within the rumen, reticulum and omasum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but in calves within the abomasum, duodenum, jejunum, and colon (P\u0026thinsp;=\u0026thinsp;0.005 for the colon and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for the abomasum, duodenum, and jejunum) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGPR173\u003c/h2\u003e \u003cp\u003eIn calves, the rumen presented the highest concentration of GPR173, with slightly lower, yet not statistically significant, levels observed in the reticulum, omasum, and abomasum (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The lowest concentrations were found in the intestines. Significant differences in GPR173 levels were identified between the following segments: the rumen and duodenum (P\u0026thinsp;=\u0026thinsp;0.002), the rumen and jejunum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), the rumen and ileum (P\u0026thinsp;=\u0026thinsp;0.016), the rumen and colon (P\u0026thinsp;=\u0026thinsp;0.010), and the omasum and jejunum (P\u0026thinsp;=\u0026thinsp;0.023).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e In adult cattle, the omasum presented the highest concentration of GPR173, followed by the reticulum, abomasum, and duodenum, with no significant differences among them (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The remaining segments of the GIT presented the lowest levels of GPR173. Significant differences were noted between the following segments: the rumen and omasum (P\u0026thinsp;=\u0026thinsp;0.004), the reticulum and jejunum (P\u0026thinsp;=\u0026thinsp;0.005), the reticulum and ileum (P\u0026thinsp;=\u0026thinsp;0.007), the omasum and jejunum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), the omasum and ileum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the omasum and colon (P\u0026thinsp;=\u0026thinsp;0.005).\u003c/p\u003e \u003cp\u003eComparative analysis between age groups revealed a significant difference in GPR173 levels in the duodenum, with adults exhibiting higher levels than calves (P\u0026thinsp;=\u0026thinsp;0.018) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe findings from this study provide a comprehensive analysis of the immunolocalization and immunoexpression levels of PNX-14 and its receptor GPR173 across various segments of the bovine GIT in both calves and adult cattle. The differential expression patterns observed suggest potentially significant functional roles for these molecules in bovine GIT physiology and possibly reveal age-related differences in their regulatory mechanisms. Additionally, the methodologies employed in this study, including immunohistochemistry (IHC) and ELISA, provide a framework for confirming the localization and immunoexpression levels of PNX-14 and GPR173, lending credibility to the conclusions drawn (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe levels of PNX-14 were higher in calves compared to adults in all segments except the forestomachs, where adults presented greater concentrations. This finding provides valuable insights into the peptide's potential roles, as the rumen, reticulum, and omasum have limited activity in calves and gradually develop their functions with the introduction of solid feed (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This pattern suggests that PNX-14 may play a significant role in food processing. In support of this hypothesis, PNX has also been detected in premetazoans, where it is believed to influence food intake (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). However, this does not preclude the function of the peptide in the forestomachs of calves, as its levels in the rumen and reticulum were still higher than in the intestines. These findings indicate that PNX-14 may contribute to the early development of these compartments. The eight-month-old calves in this study had transitioned from monogastric to polygastric digestion, suggesting that their forestomachs were functional but not yet fully mature compared with those of adults. This transition is reflected in the higher levels of PNX-14 observed in the rumen, reticulum, and omasum of adult cattle. These compartments play crucial roles in microbial fermentation and nutrient absorption, processes that require mucosal protection and regulation. The increased PNX-14 levels in these compartments in adults likely support these functions, helping to maintain mucosal integrity and manage the inflammatory responses associated with microbial activity. The protective effects of PNX-14 against gastrointestinal stressors highlighted by Zandeh-Rahimi et al. in 2022 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) demonstrated its ability to significantly reduce macroscopic duodenal lesions, lower the serum levels of proinflammatory cytokines, and decrease the activity of oxidative stress markers such as malondialdehyde and myeloperoxidase in rats. However, the lower levels of PNX-14 detected in the bovine GIT compared with the higher concentrations administered extrinsically in Zandeh-Rahimi et al.'s study suggest that these protective benefits might not be as pronounced in cattle. The physiological conditions in bovines likely involve much lower concentrations of PNX-14, which may not be sufficient to elicit the same extent of protective effects observed in the rat model. This disparity indicates that while PNX-14 could offer some benefits in mitigating gastrointestinal stressors, its impact on cattle may be limited due to its lower endogenous levels. Consequently, the protective role of PNX-14 in the bovine GIT may be less significant, necessitating further research to explore the specific conditions under which PNX-14 might exert its beneficial effects.\u003c/p\u003e \u003cp\u003eThe higher PNX-14 levels in the abomasum of calves suggest that PNX-14 may play a role in protecting the mucosal lining and modulating inflammation in this critical digestive compartment as calves transition from milk to solid feed (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The difference in the level of PNX was also noticeable in the IHC study. The abomasum of calves was characterized by the presence of PNX-IR within parietal cells. It can therefore be speculated that in young cattle, PNX somehow aids in regulating gastric acid secretion and, consequently, supports digestive and antimicrobial protective functions (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are no studies available on the concentration of PNX in the GIT in cattle. However, a study conducted in rats by discoverers of PNX demonstrated the highest levels in the stomach, followed by the jejunum, duodenum, ileum, and colon (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Furthermore, the protein levels obtained in the present study are consistent with those reported in a previous study assessing the mRNA levels of the precursor SMIM20, as \u003cem\u003eSMIM20\u003c/em\u003e mRNA was most abundant in the rumen and reticulum (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Notably, the difference between mRNA and protein levels is highlighted by the lack of statistically significant differences in mRNA levels between adult individuals and calves (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImmunolocalization of PNX-14 observed exclusively in the mucosa of all sections of the GIT in relatively small amounts (approximately 1 IR cell per 100 cells in most cases) may also support the notion that it does not play significant roles in the cattle GIT. On the other hand, while the levels of PNX-14 detected in the bovine GIT are relatively low, this does not necessarily imply that its role is insignificant. The small amounts of PNX-14 could be due to the small size of the peptide, but the possibility that even low concentrations can exert significant biological effects is probable. For example, interleukin-6, a protein known for its crucial role in inflammation and immune responses, also exists at low concentrations but has profound impacts on physiological processes (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). This, in turn, may support the possibility that even small amounts of PNX-14 could play important roles in maintaining gut health and regulating various functions within the GIT. Another possible explanation could be the release of PNX under specific conditions (as referenced in previous studies, such as during stress or inflammation), which were not met in this study because the animals were healthy (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Furthermore, since PNX is an orexigenic peptide that inhibits appetite (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), it is possible that its overproduction occurs postprandially, which we could not assess owing to the fasting of the animals before slaughter.\u003c/p\u003e \u003cp\u003eAn immunolocalization study of PNX-14 in the rat GIT demonstrated the presence of PNX-14 between the intestinal glands and the lamina propria of the mucosa but not in epithelial cells. Interestingly, no IR was detected in the stomach or colon (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Our study differs from the aforementioned research, as we observed an IR in all segments of the GIT, from the stomach to the colon. Additionally, we noted PNX-IR in the intestinal glands, as well as an IR between the glands in the colon. This particular localization in the lamina propria suggests that PNX-14 may be potentially involved in immune functions, as it is located in areas populated by cells usually responsible for the immune response (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Given the observed discrepancies in the research, further exploration of this topic is clearly needed. Conducting additional analyses involving other species will be essential to deepen our understanding and address the existing gaps in knowledge.\u003c/p\u003e \u003cp\u003eGPR173 showed distinct localization patterns throughout the entire GIT, particularly in the nerve fibres within both the submucosal and muscular plexuses. The prominent localization of GPR173 in neural tissues indicates that it plays a significant role in modulating gut motility and neural signalling. The uneven reactions of GPR173 in nerve fibres suggest that its activity might be finely tuned to specific physiological conditions, contributing to the adaptive responses of the GIT to various stimuli. Additionally, the reaction was incidentally present in individual neurons of the intestines, indicating nonuniform localization of the receptor. This finding aligns with the established roles of GPCRs in the enteric nervous system, where they modulate various aspects of gut motility and secretion (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Additionally, GPR173 was observed in epithelial cells and blood vessels throughout the entire GIT, suggesting its involvement in epithelial and vascular functions. In all segments of the intestine, IR was also noted in the lamina propria, possibly in immune cells. The granular presence of the receptor in these areas, coupled with the low levels of PNX-14, implies that GPR173 might interact with other ligands to fulfil its roles and the fact that GPR173 levels were much higher than PNX-14 levels (PNX-14 in pg/mg of total protein, whereas GPR173 in ng/mg of total protein) further support this notion. Notably, He et al. postulated that GPR173 is a receptor for CCK, which is known to be an important gastrointestinal hormone (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). This could be a plausible explanation for the widespread distribution of GPR173 observed in our study.\u003c/p\u003e \u003cp\u003eThe significant differences in GPR173 levels between segments such as the rumen and intestines highlight the receptor\u0026rsquo;s segment-specific roles, suggesting broader mechanisms of nutrient regulation and absorption in the bovine GIT. The age-related differences in GPR173 levels, particularly the higher levels in the duodenum of adults than in those of calves, suggest developmental changes in digestive physiology and nutrient absorption efficiency. This differential expression indicates a potential adaptation to increased metabolic demands in adults. The localization of GPR173 aligns with the observations of Aleti et al., indicating that GPCR family receptors might mediate interactions between microbial metabolites and human cells and could be responsible for regulating the gut\u0026ndash;immune\u0026ndash;brain axis (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are no other studies describing the immunolocalization of GPR173 in the GIT. However, our study on the expression of \u003cem\u003eGPR173\u003c/em\u003e mRNA in the bovine GIT revealed somewhat different expression patterns compared with the protein concentration. For both mRNA and protein, the highest levels were noted in the omasum. However, the mRNA levels were much more variable between different sections compared to the protein levels, which were relatively consistent across various sections of the GIT (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur observation that PNX-14 and GPR173 are consistently present throughout the GIT supports their peripheral roles, possibly distinct from their central effects on food intake regulation. These findings align with those of Schalla et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), who reported that PNX-14 does not cross the blood‒brain barrier in vitro, potentially indicating its localized action in peripheral tissues. This, in turn, may support the notion that PNX-14 might not function as a gut\u0026ndash;brain peptide, with its effects being confined to peripheral tissues rather than influencing central nervous system processes. Moreover, the documented challenges of intraperitoneal administration to induce the expected orexigenic effect of PNX (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and the peptide\u0026rsquo;s ability to stimulate insulin secretion (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) suggest that PNX\u0026rsquo;s functional scope extends to metabolic regulation within the GIT. Intraperitoneal administration difficulties may arise from the challenges in ensuring effective delivery and bioavailability of PNX-14, possibly due to its rapid degradation or insufficient targeting of specific receptor sites within the GIT. This suggests that PNX-14\u0026rsquo;s effects are highly context-dependent and may require precise delivery mechanisms to elicit significant biological responses. The peptide's ability to stimulate insulin secretion also points to a broader role in metabolic regulation, potentially influencing glucose homeostasis and energy balance within the GIT. This metabolic role may involve intricate interactions with other gut hormones and regulatory peptides, further underscoring the complexity of PNX-14\u0026rsquo;s functions beyond its primary sites of action.\u003c/p\u003e \u003cp\u003eOne of the limitations of this study is that PNX-14 is the shortest form of PNX, which may cause an antibody directed against PNX-14 to also bind to the longer forms of PNX. Another previously mentioned limitation is that PNX has orexigenic properties and may be released postprandially. However, only fasted animals could be used in the study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, despite the low concentration of PNX-14 in the bovine GIT, it cannot be ruled out that it may play a significant role in digestive processes or the immune response. Conversely, GPR173 is more abundant, indicating that it may interact with other ligands and contribute to GIT functions through broader mechanisms. The peripheral roles of PNX-14 and GPR173, which are distinct from their central nervous system effects, suggest localized actions within the GIT that merit further investigation. The complexities of their interactions and the context-dependent nature of their effects highlight the need for precise delivery mechanisms and a deeper understanding of their roles in bovine health and nutrition.\u003c/p\u003e \u003cp\u003eThis study fills a gap in the information concerning the localization and levels of PNX-14 and its receptor GPR173 in the GIT of domestic cattle. On the basis of these results, further research can undoubtedly be conducted in other species, as well as in domestic cattle. The high levels of PNX in forestomachs, which are unique to ruminants, may indicate that PNX has important functions in polygastric animals.\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eAnimals and tissue samples\u003c/h2\u003e \u003cp\u003eThis study involved healthy male Polish Holstein\u0026ndash;Friesian cattle, including two distinct age groups: six adult animals aged 20\u0026ndash;24 months, weighing 768\u0026thinsp;\u0026plusmn;\u0026thinsp;46 kg, and six calves aged 7\u0026ndash;8 months, weighing 218\u0026thinsp;\u0026plusmn;\u0026thinsp;23 kg. All the subjects were sourced from a single farm, ensuring uniform living conditions, diet, and environmental factors. Both groups followed a semi-intensive feeding regimen, consisting of initial grazing on pasture followed by the total mixed ration feeding method, as described by Włodarczyk et al. in 2011 (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTissue samples were collected at a local slaughterhouse. Prior to slaughter, the cattle were fasted for 18 hours. Next, tissue sections were obtained from various segments of the GIT, including the rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, and colon. Care was taken to include all layers of each GIT section in the excised samples. Immediately after collection, the tissues were rinsed with a physiological saline solution to remove any residual contents and contaminants. Part of the material designated for ELISA testing was immediately placed in liquid nitrogen and then transferred to -80\u0026deg;C, while samples intended for IHC studies were placed in buffered 4% formaldehyde.\u003c/p\u003e \u003cp\u003eAdditionally, postmortem examinations confirmed the good health of the animals and revealed no pathologies within their digestive tracts, confirming the validity of the collected samples for this study. Given that tissue collection was conducted postmortem, ethical review and approval by the Ethics Committee were not required under Polish law.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTissue processing\u003c/h2\u003e \u003cp\u003eSamples fixed for 24 h in formaldehyde were rinsed in tap water, dehydrated in an increasing ethanol series, cleared in xylene and infiltrated with paraffin. The fixed samples were then embedded in paraffin via a modular embedding center (MYR EC-350, Casa \u0026Aacute;lvarez Material Cient\u0026iacute;fico SA, Madrid, Spain) and subsequently sliced into 5 \u0026micro;m-thick sections via a rotary microtome (HM 360, Microm, Walldorf, Germany). Every fifth slice was placed on SuperFrost\u0026reg; Plus slides (Thermo Scientific, Menzel-Glaser, Braunschweig, Germany) and stored in an incubator (CG Wamed, Warsaw, Poland) at 37\u0026deg;C for 12 h.\u003c/p\u003e \u003cp\u003eFrom each frozen sample, 100 mg was weighed and homogenized in phosphate-buffered saline (PBS, 0.1 M, pH\u0026thinsp;=\u0026thinsp;7.3) at a ratio of 1:9 (tissue:PBS). The homogenates were centrifuged at 12,000 \u0026times; g for 10 minutes at 4\u0026deg;C to remove debris, and the supernatants were collected. The protein content was subsequently determined using Pierce BCA kit (Thermo Scientific, Waltham, MA, USA) and expressed in \u0026micro;g/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIHC\u003c/h2\u003e \u003cp\u003eFor this study, IHC analysis was carried out to detect PNX-14 and GPR173 in the GIT tissues of calves and adult domestic cattle. The IHC analysis followed the protocol described by Osiak-Wicha et al., (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) with additional steps utilizing xylene for dewaxing the slides and rehydration in a descending concentration of ethyl alcohol. During the first day, using the sodium-citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH\u0026thinsp;=\u0026thinsp;6.0), heat-induced epitope retrieval was performed (8 min at 80\u0026deg;C, multicooker RMC-PM381-E Redmond, China), followed by a 10-minute incubation in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Next, after blocking in UltraVision Protein Block (5 min; Thermo Scientific, Waltham, MA, USA), an overnight incubation in 4\u0026deg;C with primary antibodies directed against PNX-14 and GPR173 was carried out (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Only one primary antibody per section was used. The next day, the sections were incubated with a BrightVision two-step detection system of poly-HRP-anti-Ms/Rb IgG (ImmunoLogic WellMed B.V., Duiven, Netherlands; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) for a total of 45 min, and immunostaining was performed with 3,3\u0026prime;-diaminobenzidine (DAB substrate kit; ab64238; Abcam, Cambridge, UK). PBS was used to rinse the slides between each step. After DAB staining, the sections were washed with distilled water, counterstained with Meyer\u0026rsquo;s hematoxylin (Patho, Mar-Four, Konstantyn\u0026oacute;w Ł\u0026oacute;dzki, Poland), washed with tap water for 10 minutes, dehydrated with ascending concentrations of ethyl alcohol, cleared with xylene, cover-slipped with Shandon Consul-Mount (Thermo Scientific, Waltham, MA, USA), and dried in an incubator (CG Wamed, Warsaw, Poland) at 37\u0026deg;C for 12 h. The specificity of the antibodies was evaluated through two methods: a negative control, where the primary antibodies were substituted with the antibody diluent, and a preabsorption experiment, where the primary antibodies were mixed with an excess of the target synthetic protein before incubation. No positive IR was observed in any of the control sections.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimary and secondary antibodies used in the study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eHost\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eCatalog number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eManufacturer\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimary antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnti \u0026ndash; phoenixin-14 amide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003erabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eH-079-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003ePhoenix Pharmaceuticals, Burlingame, CA, US\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnti-GPR173\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003erabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003ePA5-33664\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003e8.4 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eThermo Scientific, Menzel-Glaser, Braunschweig, Germany\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSecondary antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eAnti-mouse/Anti-rabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003egoat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eDPVB-HRP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRTU\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eImmunoLogic, Duiven, Netherlands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eRTU = Ready To Use\u003c/p\u003e \u003cp\u003eThe stained slides were examined under a light microscope (BX-51 DSU, Olympus, Tokyo, Japan) equipped with a digital color camera (DP-70, Olympus, Tokyo, Japan). High-resolution digital photographs were taken using Cell^M 2.3 software (Olympus cellSens Standard) under consistent lighting conditions and uniform settings for brightness and contrast by a single individual.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003ePNX-14 and GPR173 levels were determined in duplicate using commercially available kits (EH4521, FineTest, Wuhan, China \u0026ndash; PNX-14; MBS9330587, MyBioSource, San Diego, CA, USA \u0026ndash; GPR173) according to the manufacturer's protocol. The absorbance values were measured using the Epoch Microplate Spectrophotometer (BioTek, Winooski, VT, USA). The intra-assay coefficient of variation did not exceed 8% in either measurement, and the sensitivity of the kits was 0.938 pg/ml for PNX-14 and 0.1 ng/ml for GPR173.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe concentration of the target analyte in each sample was normalized to the total protein content. The results are expressed as pg or ng of target analyte per mg of total protein (pg/mg protein for PNX-14 and ng/mg protein for GPR173). The average results for each sample were analysed using two-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s HSD post hoc test. Homogeneity of variance was tested using Levene's test. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All the statistical analyses were conducted using GraphPad Prism, version 9.5.0 for Windows (GraphPad Software, San Diego, CA, US; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.graphpad.com\" target=\"_blank\"\u003ewww.graphpad.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.graphpad.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed on June 10, 2024).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePNX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephoenixin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSMIM20\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esmall integral membrane protein 20\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGIT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egastrointestinal tract\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eenzyme\u0026ndash;linked immunosorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehypothalamic‒pituitary‒gonadal\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSREB3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esuper\u0026ndash;conserved receptor expressed in the brain 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eG protein\u0026ndash;coupled receptors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCCK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echolecystokinin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmunoreactivity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate\u0026ndash;buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Polish law, since all tissue collection procedures were conducted post-mortem, ethical review and approval from the Ethics Committee for this study were not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by project no. WKN/MN-1/WET/22, provided by the University of Life Sciences in Lublin, Poland, for Katarzyna Kras.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.K.: conceptualization, study design, methodology, writing\u0026mdash;original draft preparation, data analysis, visualization; C.O.-W.: statistical analysis of data, writing\u0026mdash;reviewing and editing; M.B.A.: conceptualization, writing\u0026mdash;reviewing and editing, supervision. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express sincere gratitude to Professor Ewa Tomaszewska for her invaluable substantive support throughout the course of this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYa\u0026ntilde;ez-Guerra LA, Thiel D, J\u0026eacute;kely G. Premetazoan Origin of Neuropeptide Signaling. Mol Biol Evol. 2022;39(4):msac051.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYosten GLC, Lyu RM, Hsueh AJW, Avsian-Kretchmer O, Chang JK, Tullock CW, et al. A novel reproductive peptide, phoenixin. J Neuroendocrinol. 2013;25(2):206\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBillert M, Rak A, Nowak KW, Skrzypski M. Phoenixin: More than Reproductive Peptide. Int J Mol Sci. 2020;21(21):8378.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrinz P, Scharner S, Friedrich T, Schalla M, Goebel-Stengel M, Rose M, et al. Central and peripheral expression sites of phoenixin-14 immunoreactivity in rats. Biochem Biophys Res Commun. 2017;493(1):195\u0026ndash;201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchalla MA, Stengel A. Phoenixin-A Pleiotropic Gut-Brain Peptide. Int J Mol Sci. 2018;19(6):1726.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang H, Zhao Q, Lv S, Ji X. Regulation and physiological functions of phoenixin. Front Mol Biosci. 2022;9:956500.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcilwraith EK, Belsham DD. Phoenixin: uncovering its receptor, signaling and functions. Acta Pharmacol Sin. 2018;39(5):774\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZandeh-Rahimi Y, Panahi N, Hesaraki S, Shirazi-Beheshtiha SH. Protective Effects of Phoenixin-14 Peptide in the Indomethacin-Induced Duodenal Ulcer: An Experimental Study. Int J Pept Res Ther. 2022;28(1):43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKupcova I, Danisovic L, Grgac I, Harsanyi S. Anxiety and Depression: What Do We Know of Neuropeptides? Behav Sci Basel Switz. 2022;12(8):262.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcIlwraith EK, Zhang N, Belsham DD. The Regulation of Phoenixin: A Fascinating Multidimensional Peptide. J Endocr Soc. 2022;6(2):bvab192.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe L, Shi H, Zhang G, Peng Y, Ghosh A, Zhang M, et al. A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition. J Neurosci Off J Soc Neurosci. 2023;43(13):2305\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKras K, Ropka-Molik K, Muszyński S, Arciszewski MB. Expression of Genes Encoding Selected Orexigenic and Anorexigenic Peptides and Their Receptors in the Organs of the Gastrointestinal Tract of Calves and Adult Domestic Cattle (\u003cem\u003eBos taurus taurus\u003c/em\u003e). Int J Mol Sci. 2023;25(1):533.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGovil K, Yadav D, Patil A, Nayak S, Baghel R, Yadav P, et al. Feeding management for early rumen development in calves. J Entomol Zool Stud. 2017;5(3):1132\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePisoni L, Relling AE. The effects of supplementing yeast fermentation products on gut permeability, hormone concentration, and growth in newborn dairy calves. Transl Anim Sci. 2020;4(2):809\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEngevik AC, Kaji I, Goldenring JR. The Physiology of the Gastric Parietal Cell. Physiol Rev. 2020;100(2):573\u0026ndash;602.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomaszewska E, Świątkiewicz M, Muszyński S, Donaldson J, Ropka-Molik K, Arciszewski MB, et al. Repetitive Cerulein-Induced Chronic Pancreatitis in Growing Pigs-A Pilot Study. Int J Mol Sci. 2023;24(9):7715.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriedrich T, Stengel A. Current state of phoenixin-the implications of the pleiotropic peptide in stress and its potential as a therapeutic target. Front Pharmacol. 2023;14:1076800.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchalla MA, Oerter S, Cubukova A, Metzger M, Appelt-Menzel A, Stengel A. Locked Out: Phoenixin-14 Does Not Cross a Stem-Cell-Derived Blood\u0026ndash;Brain Barrier Model. Brain Sci. 2023;13(7):980.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontalban-Arques A, Chaparro M, Gisbert JP, Bernardo D. The Innate Immune System in the Gastrointestinal Tract: Role of Intraepithelial Lymphocytes and Lamina Propria Innate Lymphoid Cells in Intestinal Inflammation. Inflamm Bowel Dis. 2018;24(8):1649\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeinberg ZY, Puthenveedu MA. Regulation of G protein-coupled receptor signaling by plasma membrane organization and endocytosis. Traffic Cph Den. 2019;20(2):121\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAleti G, Troyer EA, Hong S. G protein-coupled receptors: A target for microbial metabolites and a mechanistic link to microbiome-immune-brain interactions. Brain Behav Immun - Health. 2023;32:100671.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchalla M, Prinz P, Friedrich T, Scharner S, Kobelt P, Goebel-Stengel M, et al. Phoenixin-14 injected intracerebroventricularly but not intraperitoneally stimulates food intake in rats. Peptides. 2017;96:53\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWłodarczyk R, Budvytis M. Proper nutrition for high yielding cows - how to fully utilize their production potential. Vet Life. 2011;86:771\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsiak-Wicha C, Muszyński S, Tomaszewska E, Kras K, Ropka-Molik K, Zhyla M, et al. Gene Expression Level and Immunohistochemical Localization of Cannabinoid and Cannabinoid-Related Receptors in The Small Intestine of Holstein Bulls (\u003cem\u003eBos taurus taurus\u003c/em\u003e). Ann Anim Sci. 2024;24(3):779\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cattle, developmental metabolism, GPCR, PNX-14, polygastric animals, ruminants, SMIM20","lastPublishedDoi":"10.21203/rs.3.rs-4852060/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4852060/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhoenixin (PNX), an ancient but newly discovered neuropeptide, is involved in various physiological processes, such as food intake, cardiovascular functions, reproductive functions, and stress regulation. The peptide is derived from the precursor protein small integral membrane protein 20 (SMIM20) and acts through the GPR173 receptor. Due to its relatively recent discovery in 2013, there is a gap in research regarding its localization in specific organs. There are no data in the literature concerning its location and level in the gastrointestinal tract (GIT) of domestic cattle, which are among the world's main livestock animals. Due to the fact that PNX exhibits a highly conserved structure across species, it is likely that it performs key functions in the body. Therefore, this study aimed to investigate the immunolocalization and immunoexpression levels of PNX-14 and GPR173 in the GIT segments of calves and adult cattle. Study material, including GIT sections of two age groups, adults and calves of domestic cattle (n\u0026thinsp;=\u0026thinsp;6), was obtained from a slaughterhouse. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemical (IHC) analyses were performed. Analyses revealed low levels of PNX-14 in the GIT of both age groups, with localization restricted to epithelial cells across all examined GIT segments. The highest levels were observed in the rumen and reticulum, higher in adults than in calves, whereas the levels in the abomasum and intestines were higher in calves than in adults. This distribution may result from the delayed development of forestomachs in calves. The higher level of GPR173 than PNX-14 and its broader distribution may suggest that PNX-14 is not the only ligand for this receptor. Overall, the results suggest that both peptides may play protective roles related to the immune response, regulate digestive and absorptive functions, and due to receptor presence in nerve fibres, may play a role in regulating GIT secretion and motility. These findings could potentially facilitate further research into the therapeutic potential of targeting PNX-14 and GPR173 in managing gastrointestinal disorders in domestic cattle and other species.\u003c/p\u003e","manuscriptTitle":"Immunolocalization and immunoexpression levels of the novel peptide phoenixin-14 and its receptor GPR173 in the gastrointestinal tract of calves and adult domestic cattle (Bos taurus taurus)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-02 19:08:50","doi":"10.21203/rs.3.rs-4852060/v1","editorialEvents":[{"type":"communityComments","content":1},{"type":"decision","content":"Revision requested","date":"2024-09-30T04:55:01+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"215261005531588856542553768536116107605","date":"2024-09-26T02:09:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-25T14:30:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273817181047065683559211558950524763755","date":"2024-09-18T17:18:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308978970745028668951524635370449749466","date":"2024-09-03T17:29:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-20T06:19:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299927943492545417766526147796878630676","date":"2024-08-11T05:49:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288298089599880917889612531669976503639","date":"2024-08-11T04:30:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-09T03:18:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-08-08T08:03:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-06T23:28:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-06T23:27:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2024-08-03T07:43:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2c366436-b639-46e9-aea3-65d73a3dff23","owner":[],"postedDate":"September 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-24T16:00:16+00:00","versionOfRecord":{"articleIdentity":"rs-4852060","link":"https://doi.org/10.1186/s12917-025-04545-x","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2025-02-18 15:57:15","publishedOnDateReadable":"February 18th, 2025"},"versionCreatedAt":"2024-09-02 19:08:50","video":"","vorDoi":"10.1186/s12917-025-04545-x","vorDoiUrl":"https://doi.org/10.1186/s12917-025-04545-x","workflowStages":[]},"version":"v1","identity":"rs-4852060","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4852060","identity":"rs-4852060","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}