by isotope dilution using13C 15N-labeled Effectsof dietary and supplementations on serotonin and extracellular (media) compartments were detennined were perfonned using R Studio; treatment and growth performanceof growmg pigs AA as an external tracer. All the statistical procedures and tendencies at P':.;0.10. Isotope ratios (IR) were and time difference were assumed at P'.S0.05 WB. Kwon1, K.J. To11chet1e1, A. Simongiovanni3, K. Syriopoufos3, A. Wessefs4 and H.H. Stein1 • 13 Area)/(Unlabeled AA Area). Samples were separated calculated for 15N and C as: (Labeled AA 1 Department ofAnimal Sciences, University oflflinois, Urbana, IL 61801, USA; 2 Animal (IC) and extracellular (EC). A fittedlinear model was by treatment and sample type: intracellular North America Inc., Chicago, ll 60631; 3Ajinomoto Animal Nutrition Europe, Paris, isotope enrichment over time. plotted as a functionof time and IR to represent France; 4Freie UniversitiitBerlin, Germany; [email protected] Results and discussion Abstract entry and exit rate of AA in primary We used isotopically labeled AA as tracers for evaluating This experiment was conducted to test the hypothesis that increase of standardized ileal digestible a relative measure of activity through AA mammary epithelial cells. The isotope ratios will be (SID) Trp:Lys is needed in diets for growing pigs if the SID Leu:Lys exceeds the requirement to tended to affect (P0.I arrangement (100,200 or 300% SID Leu:Lys and 18,23 or 28% SID Trp:Lys),and 144 growing isotopic ratio of intracellular lie did not decreased as Val concentrations increased. Further, the pigs (28.2±1.9 kg) were assigned to the diets in a randomized complete block design. Initial and findings indicate that Leu and lie respond 9 change as Val concentrations increased. Overall, our finalbody weight of pigs and daily feed provisions were recorded,and average daily gain (ADG), and NEAA extracellular concentrations. We differentlyto the competition caused by increasing Val average daily feed intake (ADFI), and gain to feed ratio were calculated. On d 0,11 and 21,blood different pools: as intracellular free AA can evaluate the uptake of AA into the BME cells in two samples were collected, and hypothalamus samples were collected on d 21. Data were analyzed using intracellular free AA pool; however,further and intracellular-bound AA.To date,we focusedon the a response surface model by removing the terms in the model that were not significant. Interactions the bound AA pool in the BME understanding of this subject would be gained by evaluating between SID Trp:Lys and SID Leu:Lys forADG, ADFI, PS, and HS were observed. For prediction will focus on the protein bound AA pool cells. An extension of this research will be conducted that of ADG,ADFI, and HS, both positive linear SID Trp:Lys and SID Leu:Lys terms were included the other AA. and analyzing the effectof bi-directional transport on in each model. However,negative linear SID Trp:Lys and positive linear SID Leu:Lys terms were observed in the model for PS. In conclusion,increasing SID Trp:Lys in diets is needed to alleviate the Acknowledgements negative impact of excess dietary Leu on growth performanceand HS concentration of growing pigs. provided byT. Pilonero and the effortsof other members The authors are gratefulfor technical help Keywords: growth pe1formance, leucine, serotonin, IIJptophan, pigs of the laboratory group,especially Grant Kawecki. Introduction References Tryptophan is an indispensable AA that is often limiting for growth in pigs fed com- and signaling in mammalian cells and organisms. Biochemical Breier,S. and A. Breier, 2017. homeostasis meal-based diets (Lewis,200 I).Tryptophan can also act as a regulator of feed intake by enhancing Journal474 (12): 1935-1963. serotonin signaling in the brain (Henry et al., 1992),because Trp is a precursor forserotonin, which K. A. Estes and A. Myers, 2018. Predicting post-absorptive Hanigan, M.D. , R.R. White, S.1.A. Apelo, M. Aguilar, is a cerebral neurotransmitter that plays an important role in appetite regulation (Zhang et al., 47: e20160417. amino acid metabolism. Revista Brasileira de Zootecnia protein and mammary 2007). Recent study indicates that excess dietary Leu can reduce synthesis of serotonin in the brain J.J. Loor, 2009. In vitro culture characterization of a Hu, H., J. Wang, D. Bu, H. Wei, L. Zhou, F. Li and (Wessels et al., 2016),which is likely a result of reduced availability ofTrp in the brain, because Leu PloS one 4(11 ): e7636. epithelial cell line from Chinese Holstein dairy cow. and Trp are both categorized as large neutral AA,and therefore,share a common uptake pathway 2000.Cellular uptake ofvaline by lactating porcine mammary Jackson, S.C. , J.M. Bryson, H. Wang andW.L. 1 lurley, across the blood-brain barrier (Barea et al., 2009). As a consequence,it is possible that an increased of Animal Science 78 (11 ): 2927-2932. tissue. Journal Wilson, concentration of dietary Trp is needed to overcome the reduction in serotonin concentrations that H. Wang, B. Nyfeler, H. Yang, M. Hild, C. Kung, C. Nicklin, P., P. Bergman, B. Zhang, E. Triantafellow, is caused by excess dietary Leu. L.C. Cantley, P.M. Finan, L.0. Murphy, 2009. Bidirectional V.E. Myer, J.P. MacKeigan, J.A. Porter, Y.K. Wang, Cell 136(3): 521-534. transport of amino acids regulates mTOR and autophagy. Material and methods

A total of 144 growing pigs were divided into 2 blocks and randomly assigned to 9 dietary treatments in a randomized complete block design. Pigs were housed in pens,and each treatment had 8 replicate pens (2 pigs per pen and 4 replicate pens per block). Nine diets based on corn,soybean meal,wheat, and barley were formulatedto contain differentlevels of standardized ilea( digestible (SID)Trp ( 18, 23 or 28% SID Trp:Lys) and SID Leu (100,200 or 300% SID Leu:Lys). The average daily gain (ADG),average daily feed intake (ADFl),and gain to feed ratio were calculated for each pen of pigs and foreach treatment group according to the initial and finalbody weight of pigs and daily feed provisions at the conclusion of the experiment. On d 0,1 1 and 21 of the experiment,blood samples were collected to analyze plasma urea nitrogen and plasma serotonin (PS),and hypothalamus samples were collected to analyze hypothalamic serotonin (HS) on the last d of the experiment.

Mario Luiz Energy and and nutrition Chizzotti (ed.) Energy and protein metabolism and nutrition 303 302 DOI 10.3920/978-90-8686-891-9_82, © Wageningen Academic Publishers 2019 Results and discussion

The reduced model was used to predict ADG, ADFI, PS, and HS (Table I). The interaction between SID Trp:Lys and SID Leu:Lys with both positive linear SID Trp:Lys and SID Leu:Lys tenns were included in each predicted model forADG, ADFI, and HS. For predicted model of PS concentration, however, the negative linear SID Trp:Lys and positive SID SID Leu:Lys tenns along with the interaction between SID Trp:Lys and SID Leu:Lys were included. Increasing SID Trp:Lys in diets is needed to alleviate the negative impact of excess dietary Leu on growth perfonnance and HS concentration of growing pigs.

Table 1. Least squares means for concentrations of plasma urea nitrogen (PUN), plasma serotonin, and cerebral serotonin of growing pigs fed diets with va1J1ing ratios between standardized ilea/ digestible (SID)Leu:Lys and SID Tlp:Lys.

SID Leu:Lys, %: 100 200 300 SEM

SID Trp:Lys, %: 18 23 28 18 23 28 18 23 28

Growth perfonnance ADG, g/d 1 867 898 852 845 869 905 750 815 777 61 ADFI, g/d2 1,675 1,724 1,630 1,657 1,656 1,720 1,519 1,584 1,506 95 G:F3 0.52 0.52 0.52 0.51 0.52 0.53 0.49 0.51 0.51 0.02 Serotonin, ng/ml Plasma4 30.6 33.2 32.4 30.7 23.8 18.6 26.6 27.4 18.6 4.4 Hypothalamus5 69.5 69.7 71.6 64.6 66.2 69.3 62.7 63.6 63.3 4.5

1 Results indicated that ADG from d Oto d 21 at different combinations of SID Trp and SID Leu could be described by the following model: 975.196 - 1.792 x SID Trp - 0.944 x SID Leu+ 0.021 x SID Trp" SID Leu (P<0.05). 2 Results indicated that ADFI from d Oto d 21 at diflerent combinations of SID Trp and SID Leu could be described by the following model: 1839.196 - 0.299 x SID Trp- 1.062 x SID Leu+ 0.016 x SID Trp x SID Leu (P<0.05). 3 Results indicated that G:F could not be predicted using SID Trp or SID Leu. 4 Results indicated that serotonin concentration in plasma at different combinations of SID Trpand SID Leu could be described by the following model: 26.844 + 0.322 x SID Trp + 0.076 x SID Leu 0.005 x SID Trp x SID Leu (P<0.05). 5 Results indicated that serotonin concentration in hypothalamus at different combinations of SID Trp and SID Leu could be described by the following model: 65.054+0.397 x SIDTrp-0.020 x SID Leu-0.001 x SIDTrp" SID Leu(P<0.05).

Acknowledgements

Financial support from Ajinomoto Animal Nutrition North America. References

Lewis, A.J., 2001. Amino acids in swine nutrition. In: Lewis, A.J. and L.L. Southern (eds.) Swine nutrition 2nd ed. CRC Press, Boca Raton, FL, USA, pp. 131-150. hang, H., J. Yin, D. Li, X. Zhou and X. Li, 2007. Tryptophan enhances ghrelin expression and secretion associated with increased foodintake and weight gain in weanling pigs. Domestic Animal Endocrinology 33: 47-61. Wessels, A.G., H. Kluge, F. Hirche,A. Kiowski, J. Bartelt, E. Corren! and G.I. Stangl, 2016. High leucine intake reduces the concentration of hypothalamic serotonin in piglets. Journalof Animal Science 94: 26-29. Barea, R., L. Brossard, N. Le Floc'h, Y. Primo! and J. van Milgen, 2009. The standardized ilea! digestible ­ to- requirement ratio may be less than fifty percent in eleven- to twenty-three-kilogram piglets. Journal of Animal Science 87: 4022-4031.

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