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

Abstract

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.

Keywords: amino acid; chicken; taste; taste receptor; umami.

Fig. 1.

Experimental procedure of the conditioned taste aversion (CTA) test. Throughout the experimental period, which included the training period, chickens were deprived of water for 6 h (from 9:00 am to 3:00 pm) and then presented water or L-amino acid solutions for 5 min. After the training period for water drinking (days 1 to 3), chickens were provided with L-amino acid solutions and administered an intraperitoneal (IP) injection of LiCl or saline on days 4 and 6. Water or L-amino acid solution intakes for 5 min were measured from days 5 to 8.

Fig. 2.

Water or 0.3 M L-Ala solution intake/bodyweight (BW) of chickens conditioned to avoid 0.3 M L-Ala solution (conditioned stimulus; CS) by LiCl or given saline (control). (A & C) Water intake/BW of chickens injected with LiCl or saline on day 5 (A) or day 7 (C). (B & D) 0.3 M L-Ala solution intake/BW of chickens injected with LiCl or saline on day 6 (B) or day 8 (D). Values represent mean±SE (saline; n=6, LiCl; n=6). * P<0.05 and ** P<0.01, unpaired t-test.

Fig. 3.

Water or 0.3 M L-Ser solution intake/BW of chickens conditioned to avoid 0.3 M L-Ser solution (CS) by LiCl or given saline. (A & C) Water intake/BW of chickens injected with LiCl or saline on day 5 (A) or day 7 (C). (B & D) 0.3 M L-Ser solution intake/BW of chickens injected with LiCl or saline on day 6 (B) or day 8 (D). Values represent mean±SE (saline; n=6, LiCl; n=7). # P<0.1 and ** P<0.01, unpaired t-test.

Fig. 4.

Water or 0.3 M L-Pro solution intake/BW of chickens conditioned to avoid 0.3 M L-Pro solution (CS) by LiCl or given saline. (A & C) Water intake/BW of chickens injected with LiCl or saline on day 5 (A) or day 7 (C). (B & D) 0.3 M L-Pro solution intake/BW of chickens injected with LiCl or saline on day 6 (B) or day 8 (D). Values represent mean±SE (saline; n=6, LiCl; n=5). * P<0.05 and *** P<0.001, unpaired t-test.

Fig. 5.

Preference ratios of L-Ala, L-Ser, and L-Pro solu-tions for chickens conditioned to avoid the corresponding L-Ala, L-Ser, or L-Pro solutions by LiCl or saline with the first conditioning (A) and second conditioning (B). Preference ratios were calculated as L-amino acid solution intake/(L-amino acid solution intake + water intake). Preference ratios with the first conditioning were calculated using L-amino acid solution intake on day 6 and water intake on day 5 (A). Preference ratios with the second conditioning were calculated using L-amino acid solution intake on day 8 and water intake on day 7 (B). Values represent mean±SE (L-Ala: saline; n=6, LiCl; n=6; L-Ser: saline; n=6, LiCl; n=7; L-Pro: saline; n=6, LiCl; n=5). * P<0.05 and ** P<0.01, unpaired t-test.

Fig. 6.

Relative mRNA levels of CALHM1 and CALHM3 were significantly higher in the palate and base of the oral cavity compared with that in the tongue tip in chickens. Values represent mean relative mRNA levels (normalized to GAPDH)±SE (n=4). Bars without a common letter differ significantly, P<0.05, paired t-test with Bonferroni correction.

Fig. 7.

Single oral administration with L-Ala solution didn't significantly affect oral mRNA tanscripts involved in umami taste sensing in chickens. (A) Experimental procedure for single oral ad mini stration of 0.3 M L-Ala solution or water. Chickens were deprived of water and food for 4 h before the experiment until 1 h after oral stimulation. The palate and duodenum were collected 1 h after administration. (B & C) Relative mRNA levels of T1R1, T1R3, TRPM5, CALHM1, and CALHM3 in the palate (B) and duodenum (C) were not significantly different between the chickens orally stimulated by L-Ala solution or water. Values represent mean relative mRNA levels (normalized to GAPDH)±SE (n=6). P>0.05, unpaired t-test.

Fig. 8.

Chronic exposure to L-Ala solution significantly downregulated oral mRNA transcripts involved in umami taste sensing in chickens. (A) Experimental procedure for chronic exposure of chickens to 0.28 M L-Ala solution or water. After ad libitum exposure to 0.28 M L-Ala or water for 1 week, the palate and duodenum were collected. (B) Relative mRNA levels of T1R1 and T1R3, but not TRPM5, CALHM1, and CALHM3 were significantly downregulated by chronic 0.28 M L-Ala stimulation in the palate. (C) Relative mRNA levels of T1R1, T1R3, TRPM5, CALHM1, and CALHM3 in the duodenum were not significantly different between the chickens stimulated by chronic 0.28 M L-Ala or water ex posure. Values represent mean relative mRNA levels (nor malized to GAPDH)±SE (n=7). # P< 0.1, * P<0.05, and ** P<0.01, unpaired t-test.