Overlapping distributions of mammalian types I, II, and III taste cell markers in chicken taste buds.

Mammalian taste buds comprise types I, II, and III taste cells, with each type having specific characteristics: glia-like supporting cells (type I), taste receptor cells (type II), and presynaptic cells (type III). In this study, to characterize the peripheral taste-sensing systems in chickens, we analyzed the distributions of the mammalian types I, II, and III taste cell markers in chicken taste buds: glutamate-aspartate transporter (GLAST) for type I; taste receptor type 1 members 1 and 3 (T1R1 and T1R3), taste receptor type 2 member 7 (T2R7), and α-gustducin for type II; and synaptosomal protein 25 (SNAP25) and neural cell adhesion molecule (NCAM) for type III. We found that most GLAST+ taste cells expressed α-gustducin and SNAP25 and that high percentages of T1R3+ or α-gustducin+ taste cells expressed SNAP25 and NCAM. These results demonstrated a unique subset of chicken taste cells expressing multiple taste cell type marker proteins. Taken together, these results provide new insights into the taste-sensing mechanisms in vertebrate taste buds.

Intragastric administration of AMG517, a TRPV1 antagonist, enhanced activity-dependent energy metabolism via capsaicin-sensitive sensory nerves in mice.

Transient receptor potential vanilloid 1 (TRPV1), a nociceptive cation channel, is known to play roles in regulating the energy metabolism (EM) of the whole body. We previously reported that TRPV1 antagonists such as AMG517 enhanced EM in mice, however, these mechanisms remain unclear. The aim of this study was to explore the mechanisms underlying the enhancement of EM by AMG517, a selective TRPV1 antagonist, in mice. Respiratory gas analysis indicated that intragastric administration of AMG517 enhanced EM along with increasing locomotor activity in mice. Next, to clarify the possible involvement with afferent sensory nerves, including the vagus, we desensitized the capsaicin-sensitive sensory nerves of mice by systemic capsaicin treatment. In the desensitized mice, intragastric administration of AMG517 did not change EM and locomotor activity. Therefore, this study indicated that intragastric administration of AMG517 enhanced EM and increased locomotor activity via capsaicin-sensitive sensory nerves, including vagal afferents in mice.

Differences in the acidic sensitivity of transient receptor potential vanilloid 1 (TRPV1) between chickens and mice.

Chickens, one of the most important industrial animals, are a biological animal model. Here we focused on the transient receptor potential vanilloid 1 (TRPV1) to understand the pain system for acidic stimuli in chickens compared with mice. By using a whole-cell patch clamp system, we confirmed that acidic stimuli activate both chicken TRPV1 (cTRPV1) and mouse TRPV1 (mTRPV1), but the peak current of cTRPV1 is lower than that of mTRPV1, and it is difficult to desensitize cTRPV1 with an acidic stimulus compared to mTRPV1. Since the C-terminal of the calmodulin (CaM) binding site in TRPV1 was reported as one of the important structures for TRPV1 desensitization, we made chimeric cTRPV1 in which the CaM binding site of chicken is changed to that of mouse (cTRPV1-mCaM). We also compared the acidic responses of native chicken dorsal root ganglion (DRG) cells with that of mouse DRG cells. The TRPV1-mCaM results showed that the desensitization of mutant cTRPV1 was similar to that of mTRPV1, and that the basal activities of mutant cTRPV1 were significantly higher than those of cTRPV1. It was also difficult to desensitize the chicken DRG cells with an acidic stimulus, unlike the mouse DRG cells. These results suggest that there are differences in the pain transduction systems for acidic stimuli between chickens and mice that are caused by the dysfunction of the C-terminal CaM biding site of cTRPV1. These results imply that chickens repeatedly feel weak pain from an acidic stimulus, without desensitization.

Bitter taste receptor T2R7 and umami taste receptor subunit T1R1 are expressed highly in Vimentin-negative taste bud cells in chickens.

In the mammalian taste system, the taste receptor type 2 (T2R) family mediates bitter taste, and the taste receptor type 1 (T1R) family mediates sweet and umami tastes (the heterodimer of T1R2/T1R3 forms the sweet taste receptor, and the heterodimer of T1R1/T1R3 forms the umami taste receptor). In the chicken genome, bitter (T2R1, T2R2, and T2R7) and umami (T1R1 and T1R3) taste receptor genes have been found. However, the localization of these taste receptors in the taste buds of chickens has not been elucidated. In the present study, we demonstrated that the bitter taste receptor T2R7 and the umami taste receptor subunit T1R1 were expressed specifically in the taste buds of chickens labeled by Vimentin, a molecular marker for chicken taste buds. We analyzed the distributions of T2R7 and T1R1 on the oral epithelial sheets of chickens and among 3 different oral tissues of chickens: the palate, the base of the oral cavity, and the posterior tongue. We found that the distribution patterns and numbers were similar between taste bud clusters expressing these receptors and those expressing Vimentin. These results indicated broad distributions of T2R7 and T1R1 in the gustatory tissues of the chicken oral cavity. In addition, 3D-reconstructed images clearly revealed that high levels of T2R7 and T1R1 were expressed in Vimentin-negative taste bud cells. Taken together, the present results indicated the presence of bitter and umami sensing systems in the taste buds of chickens, and broad distribution of T2R7 and T1R1 in the chicken oral cavity.

Oral lipase activities and fat-taste receptors for fat-taste sensing in chickens.

It has been reported that a functional fat-taste receptor, GPR120, is present in chicken oral tissues, and that chickens can detect fat taste in a behavioral test. However, although triglycerides need to be digested to free fatty acids to be recognized by fat-taste receptors such as GPR120, it remains unknown whether lipase activities exist in chicken oral tissues. To examine this question, we first cloned another fat-taste receptor candidate gene, CD36, from the chicken palate. Then, using RT-PCR, we determined that GPR120 and CD36 were broadly expressed in chicken oral and gastrointestinal tissues. Also by RT-PCR, we confirmed that several lipase genes were expressed in both oral and gastrointestinal tissues. Finally, we analyzed the lipase activities of oral tissues by using a fluorogenic triglyceride analog as a lipase substrate. We found there are functional lipases in oral tissues as well as in the stomach and pancreas. These results suggested that chickens have a basic fat-taste reception system that incorporates a triglycerides/oral-lipases/free fatty acids/GPR120 axis and CD36 axis.

Identification of functional bitter taste receptors and their antagonist in chickens.

Elucidation of the taste sense of chickens is important not only for the development of chicken feedstuffs for the chicken industry but also to help clarify the evolution of the taste sense among animals. There are three putative chicken bitter taste receptors, chicken T2R1 (cT2R1), cT2R2 and cT2R7, which were identified using genome information and cell-based assays. Previously, we have shown that cT2R1 is a functional bitter taste receptor through both cell-based assays and behavioral tests. In this study, therefore, we focused on the sensitivities of the other two bitter receptors, cT2R2 and cT2R7, by using their agonists in behavioral tests. We tested three agonists of cT2R2 and three agonists of cT2R7. In a 10-min drinking study, the intakes of cT2R2 agonist solutions were not different from that of water. On the other hand, the intakes of cT2R7 agonist solutions were significantly lower compared to water. In addition, we constructed cT2R1-and cT2R7-expressing cells in order to search for an antagonist for these functional bitter taste receptors. By using Ca2+ imaging methods, we found that 6-methoxyflavanone (6-meth) can inhibit the activities of both cT2R1 and cT2R7. Moreover, 6-meth also inhibited the reduction of the intake of bitter solutions containing cT2R1 or cT2R7 agonists in behavioral tests. Taken together, these results suggested that cT2R7 is a functional bitter taste receptor like cT2R1, but that cT2R2 is not, and that 6-meth is an antagonist for these two functional chicken bitter taste receptors. This is the first identification of an antagonist of chicken bitter receptors.

The role of G-protein-coupled receptor 120 in fatty acids sensing in chicken oral tissues.

Clarification of the mechanism of chickens' taste sense will provide meaningful information for creating and improving new feedstuff for chickens, because the character of taste receptors in oral tissues affects feeding behavior in animals. Although fatty acids are partly recognized via G-protein coupled receptor 120 (GPR120) for fat taste in mammalian oral tissues, the fat taste receptor of chickens has not been elucidated. Here we cloned chicken GPR120 (cGPR120) from the chicken palate, which contains taste buds. By using Ca(2+) imaging methods, we identified oleic acid and linoleic acid as cGPR120 agonists. Interestingly, in a behavioral study the chickens preferred corn oil-rich feed over mineral oil (control oil)-rich feed. Because corn oil contains high amounts of oleic acid and linoleic acid, this result was thought to be reasonable. Taken together, the present results suggest that cGPR120 is one of the functional fat taste receptors in chickens.

Bitter taste receptor T2R1 activities were compatible with behavioral sensitivity to bitterness in chickens.

Clarification of the mechanism of the sense of taste in chickens will provide information useful for creating and improving new feedstuffs for chickens, because the character of the taste receptors in oral tissues affects feeding behavior in animals. In this study, we focused on the sensitivity to bitterness in chickens. We cloned one of the bitter taste receptors, T2R1, from the chicken palate, constructed several biosensor-cells expressing chicken T2R1 (cT2R1), and determined a highly sensitive biosensor of cT2R1 among them. By using Ca(2+) imaging methods, we identified two agonists of cT2R1, dextromethorphan (Dex) and diphenidol (Dip). Dex was a new agonist of cT2R1 that was more potent than Dip. In a behavioral drinking study, the intake volumes of solutions of these compounds were significantly lower than that of water in chickens. These aversive concentrations were identical to the concentrations that could activate cT2R1 in a cell-based assay. These results suggest that the cT2R1 activities induced by these agonists are linked to behavioral sensitivity to bitterness in chickens.

Expressions of multiple umami taste receptors in oral and gastrointestinal tissues, and umami taste synergism in chickens.

Umami taste is one of the five basic taste qualities, along with sweet, bitter, sour, and salty, and is elicited by some l-amino acids and their salts, including monopotassium l-glutamate (MPG). The unique characteristic of umami taste is that it is synergistically enhanced by 5'-ribonucleotides such as inosine 5'-monophosphate (IMP). Unlike the other four basic taste qualities, the presence of umami taste sense in avian species is not fully understood. In this study, we demonstrated the expression of multiple umami taste receptor candidates in oral and gastrointestinal tract tissues in chickens using RT-PCR analysis. We first showed the metabotropic glutamate receptors (mGluRs) expressed in these tissues. Furthermore, we examined the preference for umami taste in chickens, focusing on the synergistic effect of umami taste as determined by the two-feed choice test. We concluded that chickens preferred feed containing both added MPG and added IMP over feeds containing either added MPG or added IMP alone and over the control feed. These results suggest that the umami taste sense and synergism are conserved in chickens.

Sex differences in COL1A1 Expression and Collagen Content in Skeletal Muscle of Mature and Juvenile Shamo Chickens.

Collagen content is an important parameter affecting meat consistency. Sex differences in collagen were therefore studied in mature and juvenile Shamo chickens. The pectoral (PT), lateral iliotibial (ITL), medial part of puboischiofemoral (PIF), and lateral part of gastrocnemius (GCL) muscles were weighed, and their COL1A1 expression levels and total collagen content were analyzed. Body and muscle weights were significantly higher in males than in females of all ages. Muscle/body weight ratios were also higher in mature males than in females, but this difference was not observed in juveniles. In mature chickens, COL1A1 expression was higher in the PIF and GCL muscles; this was not the case in juvenile chicken muscles. Sex differences in collagen content were observed only in the ITLs of mature chickens. A positive correlation between muscle weight and intramuscular collagen content was found for PT and GCL, but not for ITL and PIF, muscles. These results suggest that the sex difference in intramuscular collagen content only occurs in specific muscles and that COL1A1 expression is not necessarily related to collagen content in mature chickens. Factors that determine the intramuscular collagen content likely differ by muscle type.

Expression and localization of the neuropeptide Y-Y4 receptor in the chick spleen: mRNA upregulation by high ambient temperature.

High ambient temperatures (HT) can increase diencephalic neuropeptide Y (NPY) expression, and central injection of NPY attenuates heat stress responses while inducing an antioxidative state in the chick spleen. However, there is a lack of knowledge about NPY receptor expression, and its regulation by HT, in the chick spleen. In the current study, male chicks were used to measure the expression of NPY receptors in the spleen and other immune organs under acute (30 vs. 40 ± 1°C for 3 h) or chronic (30 vs. 40 ± 1°C for 3 h/day for 3 days) exposure to HT and in response to central injection of NPY (47 pmol, 188 pmol, or 1 nmol). We found that NPY-Y4 receptor mRNA was expressed in the spleen, but not in other immune organs studied. Immunofluorescence staining revealed that NPY-Y4 receptors were localized in the splenic pulp. Furthermore, NPY-Y4 receptor mRNA increased in the chick spleen under both acute and chronic exposure to HT. Central NPY at two dose levels (47 and 188 pmol) and a higher dose (1 nmol) did not increase splenic NPY-Y4 receptor mRNA expression or splenic epinephrine under HT (35 ± 1°C), and significantly increased 3-methoxy-4-hydroxyphenylglycol (MHPG) concentrations under HT (40 ± 1°C). In conclusion, increased expression of NPY-Y4 receptor mRNA in the spleen under HT suggest that Y4 receptor may play physiological roles in response to HT in male chicks.

Collagen Content and Collagen Fiber Architecture in the Skin of Shamo Chicken, a Japanese Game Fowl.

Collagen content and collagen fiber architecture in the skin of Shamo chickens were compared between sexes and body parts. Cervical, thoracic, dorsal, femoral, and crural skin samples were collected and their collagen content was analyzed. Collagen fiber specimens were prepared for scanning electron microscopy using the cell maceration method with a NaOH solution. Sex differences in collagen content were only observed in the femoral skin of mature chickens, but not in 10-week-old chicks. The difference in collagen content between body parts was obvious; femoral and crural skin had higher collagen content than those of other parts in both sexes. Scanning electron microscopy indicated that the collagen fiber architecture was quite different between the superficial and deep layers in the dermis, with the former consisting of loosely tangled band-like collagen fibers, and the latter composed of thick and dense layers of collagen bundles in a parallel arrangement. The width of collagen fibers in the superficial layer of the dermis differed between sexes in the dorsal, femoral, and crural skin. From these results, it is likely that the difference in collagen content in the femoral skin is not due to sex hormones but other factors, such as mechanical stimulation in daily activity. Additionally, collagen fiber width in the superficial layer is likely related to the difference in collagen content between sexes and between body parts.

Effects of different coating materials on the morphological characteristics of chicken adenohypophyseal folliculo-stellate cells in vitro.

Chicken adenohypophyseal cells were cultured in plates coated with different materials, and their morphologies were examined to confirm the characteristics of chicken folliculo-stellate (FS) cells in vitro. The adenohypophyseal cells were dispersed with a collagenase/trypsin mixture in media and seeded in plates coated in either poly L-lysine (PLL), collagen, or laminin. After 7 days of culture, the cells were fixed and immunocytochemistry was performed. 5-Bromo-2'-deoxyuridine incorporation test indicated that the proliferation activity of the culture cells was different based on the coating materials, and it was higher in the collagen-coated plate than two other coating materials. Fluorescence immunocytochemistry was also performed using mixed antibodies against growth hormone, prolactin, luteinizing hormone ß-subunit, basic cytokeratin (bCK), and S100B. The culture cells on the PLL- and laminin-coated surfaces were round or oval in shape, and bCK-immunopositive FS cells were morphologically indistinguishable from endocrine cells. In the collagen-coated plate, many endocrine cells were round or oval in shape, but FS cells displayed a larger and flattened morphology. S100B-immunoreactions were localized in the nuclei of bCK-immunopositive FS cells. These results suggest that culturing the chicken adenohypophyseal cells in the collagen-coated plate enables the distinction of FS cells from endocrine cells.

Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite cell activation in response to mechanical stretch.

When skeletal muscle is stretched or injured, satellite cells, resident myogenic stem cells positioned beneath the basal lamina of mature muscle fibers, are activated to enter the cell cycle. This signaling pathway is a cascade of events including calcium-calmodulin formation, nitric oxide (NO) radical production by NO synthase, matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the receptor c-met, as demonstrated by assays of primary cultures and in vivo experiments. Here, we add evidence that two ion channels, the mechanosensitive cation channel (MS channel) and the long-lasting-type voltage-gated calcium-ion channel (L-VGC channel), mediate the influx of extracellular calcium ions in response to cyclic stretch in satellite cell cultures. When applied to 1-h stretch cultures with individual inhibitors for MS and L-VGC channels (GsMTx-4 and nifedipine, respectively) or with a less specific inhibitor (gadolinium chloride, Gd), satellite cell activation and upstream HGF release were abolished, as revealed by bromodeoxyuridine-incorporation assays and Western blotting of conditioned media, respectively. The inhibition was dose dependent with a maximum at 0.1 µM (GsMTx-4), 10 µM (nifedipine), or 100 µM (Gd) and canceled by addition of HGF to the culture media; a potent inhibitor for transient-type VGC channels (NNC55-0396, 100 µM) did not show any significant inhibitory effect. The stretch response was also abolished when calcium-chelator EGTA (1.8 mM) was added to the medium, indicating the significance of extracellular free calcium ions in our present activation model. Finally, cation/calcium channel dependencies were further documented by calcium-imaging analyses on stretched cells; results clearly demonstrated that calcium ion influx was abolished by GsMTx-4, nifedipine, and EGTA. Therefore, these results provide an additional insight that calcium ions may flow in through L-VGC channels by possible coupling with adjacent MS channel gating that promotes the local depolarization of cell membranes to initiate the satellite cell activation cascade.

Hypothalamic gonadotropin-inhibitory hormone precursor mRNA is increased during depressed food intake in heat-exposed chicks.

The regulation of food intake in chickens (Gallus gallus domesticus) represents a complex homeostatic mechanism involving multiple levels of control, and regulation during high ambient temperatures (HT) is poorly understood. In this study, we examined hypothalamic mRNA expression of gonadotropin-inhibitory hormone (GnIH) to understand the effect of HT on an orexigenic neuropeptide. We examined the effects of HT (35 °C ambient temperature for 1, 24 or 48 h) on 14-day old chicks. HT significantly increased rectal temperature and suppressed food intake, and also influenced plasma metabolites. The expression of GnIH precursor mRNA in the diencephalon was significantly increased in chicks at 24-and 48 h of HT when food intake was suppressed significantly, whilst no change was observed for GnIH precursor mRNA and food intake at 1h of HT. In situ hybridization and immunocytochemistry further revealed the cellular localization of chicken GnIH precursor mRNA and its peptide in the paraventricular nucleus (PVN) in the chick hypothalamus. We examined plasma metabolites in chicks exposed to HT for 1 or 48 h and found that triacylglycerol concentration was significantly higher in HT than control chicks at 1h. Total protein, uric acid and calcium were significantly lower in HT chicks than control chicks at 48h. These results indicate that not only a reduction in food intake and alteration in plasma metabolites but also the PVN-specific expression of GnIH, an orexigenic agent, may be induced by HT. The reduced food intake at the same time as GnIH expression was increased during HT suggests that HT-induced GnIH expression may oppose HT-induced feeding suppression, rather than promote it. We suggest that the increased GnIH expression could be a consequence of the reduced food intake, and would not be a direct response to HT.

Effects of Estradiol on Expression of Estrogen Receptor and Collagen mRNAs in Chick Skin.

Skin thickness and strength differ between male and female chickens. This study aimed to clarify the effects of estradiol on the expression of estrogen receptors and collagen mRNA in chicken skin. Estradiol was administered to male chicks for 3 weeks, then cryosections of skin collected from the cervical, thoracic, dorsal, and pelvic limb regions were stained with hematoxylin and eosin, and dermal thickness was measured. Estrogen receptor and collagen mRNA expression was assessed using real-time RT-PCR, and collagen contents were determined. Estradiol did not alter dermal thickness or the collagen content of the skin from any tested region. Among the estrogen receptors, significantly more ESR1 mRNA was expressed in the thoracic skin of chicks administered with estradiol compared with vehicle (control), and in the thoracic skin compared with skin from other regions within each group. Estradiol did not affect ESR2, GPER, and COL1A1 mRNA expression. These results suggested that estradiol stimulates ESR1 expression in thoracic skin, but does not affect collagen synthesis in skin from any other region of male chicks.

Oral expressions and functional analyses of the extracellular calcium-sensing receptor (CaSR) in chicken.

In vertebrates, the extracellular calcium-sensing receptor (CaSR) plays a key role in calcium homeostasis by sensing slight changes in extracellular Ca2+. CaSR is also expressed in mammals including rodent taste cells and is involved in sensing kokumi, a rich, savory quality that enhances the intensities of salty, sweet, and umami tastes. In this study, we focused on chicken CaSR (cCaSR) since calcium is an essential nutrient that is necessary for making eggshell and for the extremely rapid initial growth of bones. First we confirmed that cCaSR is expressed in taste cells. Next we cloned the cCaSR gene from kidney and transiently transfected human embryonic kidney 293 T (HEK293T) cells with the recombinant cCaSR, or empty vector and looked for the agonists and allosteric modulators (including kokumi substances) of cCaSR by Ca2+ imaging. We found that cCaSR was activated by extracellular Ca2+ and Mg2+ in a dose dependent manner. Several L-amino acids and kokumi substances such as glutathione enhanced the response of cCaSR. In addition, NPS2143 as a negative allosteric modulator of human CaSR negatively modulated the response of cCaSR. These results suggest that cCaSR can sense extracellular Ca2+ and Mg2+ as well as positive and negative allosteric modulators. Taken together, the results imply that CaSR might be a multifunctional receptor for calcium, amino acids, and kokumi substances in chicken. The present finding that functional CaSR is expressed in the chicken oral tissues will allow us to further elucidate the physiological role of CaSR in the chickens' taste sense, and to create new feeds that will contribute to the poultry industry.

Fatty Acid Taste Receptor GPR120 Activation by Arachidonic Acid, Eicosapentaenoic Acid, and Docosahexaenoic Acid in Chickens.

It has been reported that the supplementation of chicken diet with polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) affects the qualities of eggs and meat. Previous studies have shown that a functional fatty acid taste receptor, G protein-coupled receptor 120 (GPR120), is broadly expressed in chicken oral and gastrointestinal tissues, and chickens have a gustatory perception of oleic acid, which is a chicken GPR120 agonist. The aim of this study was to elucidate the role of chicken GPR120 in response to PUFAs in chicken diets. Ca2+ imaging analyses revealed that chicken GPR120 was activated by AA, EPA, and DHA in a concentration-dependent manner. These results suggest that chickens can detect PUFAs via GPR120 in the oral and gastrointestinal tissues, implying that chickens have a gustatory perception of PUFAs.

Identification of Ligands for Chicken Transient Receptor Potential Ankyrin 1 Channel and Chemosensory Perception of Herbal Compounds in Chickens.

The pungency induced by spices and herbs plays an important role in food choice and appetite, and it is suggested that adding spices and herbs to feed as natural alternatives to antibiotics has beneficial effects in poultry farming. However, our knowledge of the chemosensory perception of herbal compounds in chickens is limited. Transient receptor potential ankyrin 1 (TRPA1) is involved in the sensory perception of various herbal compounds. Here, we performed calcium imaging and electrophysiological analyses using cells transiently expressing chicken TRPA1 (cTRPA1) and identified two novel cTRPA1 ligands-eugenol and thymol. In a behavioral assay, chickens responded to cTRPA1 ligands, including eugenol, thymol, cinnamaldehyde, carvacrol, and allyl isothiocyanate. These results provide evidence that chickens have a functional TRPA1 channel and chemosensory perception of various herbal compounds.

Conditioned Taste Aversion to L-Amino Acid Taste Stimuli and Oral Transcriptional Changes to Type 1 Taste Receptors T1R1 and T1R3 on Chronic Exposure to L-Alanine Solution in Chickens.

Elucidating taste sensing systems in chickens is an important step toward understanding poultry nutrition. Amino acid taste receptors, type 1 taste receptors 1 and 3 (T1R1 and T1R3, respectively), are expressed in chicken taste cells, and chicken T1R1/T1R3 is activated by L-alanine (L-Ala) and L-serine (L-Ser), but not by L-proline (L-Pro). However, it is not clear whether chickens have a gustatory perception of L-amino acids. Here, we found that chickens conditioned to avoid either L-Ala, L-Ser, or L-Pro solutions could successfully learn to avoid the corresponding L-amino acid solution in the conditioned taste aversion (CTA) test. Because CTA is a well-established learning paradigm generated specifically by pairing gustatory perception and gastrointestinal malaise, the present study suggests that chickens can sense L-amino acids by gustatory perception. In addition, we found that the expression of the T1R1 and T1R3 genes was significantly downregulated in response to chronic exposure to L-Ala solution, but not to acute oral stimulation. Taken together, the present study suggests that chickens have a gustatory perception of L-amino acids, and the expression of T1R1/T1R3 mRNAs in the oral cavity can be regulated by L-amino acid intake. Since chickens can detect L-Pro solutions, additional amino acid receptors, other than T1R1/T1R3, may be involved in L-amino acid taste detection in chickens.