TY - JOUR
AU - Cervantes,, Miguel
AB - Abstract Pigs exposed to heat stress (HS) increase body temperature in which can damage the intestinal epithelia and affect the absorption and availability of amino acids (AA). Protein digestion and metabolism further increase body temperature. An experiment was conducted with six pairs of pigs (of 47.3 ± 1.3 kg initial body weight) exposed to natural HS to assess the effect of substituting dietary protein-bound AA by free AA on morphology and gene expression of intestinal epithelial and serum concentration (SC) of free AA. Treatments were: high protein, 21.9% crude protein (CP) diet (HShp) and low protein, 13.5% CP diet supplemented with crystalline Lys, Thr, Met, Trp, His, Ile, Leu, Phe, and Val (HSaa). The HShp diet met or exceeded all AA requirements. The HSaa diet was formulated on the basis of ideal protein. Pigs were fed the same amount at 0700 and 1900 hours during the 21-d study. Blood samples were collected at 1700 hours (2.0 h before the evening meal), 2030 hours, and 2130 hours (1.5 and 2.5 h after the evening meal). At the end, all pigs were sacrificed to collect intestinal mucosa and a 5-cm section from each segment of the small intestine from each pig. Villi measures, expression of AA transporters (y+L and B0) in mucosa, and SC of AA were analyzed. Ambient temperature fluctuated daily from 24.5 to 42.6 °C. Weight gain and G.F were not affected by dietary treatment. Villi height tended to be larger (P ≤ 0.10) and the villi height:crypt depth ratio was higher in duodenum and jejunum of pigs fed the HSaa diet (P < 0.05). Gene expression of transporter y+L in jejunum tended to be lower (P < 0.10) and transporter B0 in the ileum was lower (P < 0.05) in HSaa pigs. Preprandial (1700 hours) SC of Arg, His, Ile, Leu, Thr, Trp, and Val was higher (P < 0.05), and Phe tended to be higher (P < 0.10) in HShp pigs. At 2030 hours (1.5 h postprandial), serum Lys, Met, and Thr were higher in the HSaa pigs (P < 0.05). At 2130 hours (2.5 h), Arg, His, Ile, Phe, and Trp were lower (P < 0.05); Met was higher (P < 0.05); and Lys tended to be higher (P < 0.10) in HSaa pigs. In conclusion, feeding HS pigs with low protein diets supplemented with free AA reduces the damage of the intestinal epithelia and seems to improve its absorption capacity, in comparison with HS pigs fed diets containing solely protein-bound AA. This information is useful to formulate diets that correct the reduced AA consumption associated with the decreased voluntary feed intake of pigs under HS. Introduction The exposure of pigs to high ambient temperature (AT) causing heat stress (HS) increases their body temperature up to 2 °C (Morales et al., 2016). In humans, HS increases blood flow to the periphery in an attempt to eliminate the increased body heat but reduces flow to internal organs such as the intestines (Ooue et al., 2007). This blood flow redirection, however, decreases the supply of oxygen and nutrients to the small intestine, increasing the death of enterocytes (Liu et al., 2009) and affecting the integrity and absorptive capacity of the intestinal epithelia in HS pigs (Pearce et al., 2014). In addition, the digestion, absorption, and metabolism of the dietary protein produce heat above the basal metabolic, which is the largest among all nutrients (Pesta and Samuel, 2014). Free amino acids (AA) do not need enzymes for digestion and, as such, do not contribute to the digestion-related production of body heat. The reduction in the dietary protein content coupled with proper supplementation of free AA, besides improving the AA profile (NRC, 2012), may lower the production of heat associated with the oxidation of excess dietary AA that occurs when high protein diets are fed. Apparently, the efficiency of AA absorption improves when both protein-bound and free AA are included in the diet (Morales et al., 2015). Thus, we hypothesized that feeding low protein-free AA-supplemented diets can reduce the intestinal epithelia damage, improving the absorption and availability of AA of HS pigs, compared with high CP diets. The serum concentration (SC) of free AA is indicative of their absorption and availability for growth in pigs (Yen et al., 2004). Thus, the present study was conducted to analyze the intestinal morphology, gene expression of AA transporters, and SC of free AA in HS pigs fed either a high protein diet or a very low protein diet supplemented with free AA. Changes in the SC of AA between the absorptive and postabsorptive phases were also analyzed. Materials and Methods Animals and general procedure The pigs used in the present experiment were cared according to the guidelines established in the Official Mexican Regulations on Animal Care (NOM-062-ZOO-1999; Ochoa, 2001). The study was conducted during summertime when the AT rises up to 45 °C with six pairs of crossbred pigs (Landrace × Hampshire × Duroc) with an initial body weight (BW) of 47.3 ± 1.3 kg; in each pair, the pigs were of same sex and litter and similar BW. Pigs were individually housed in raised floor metabolism pens (1.2 m wide, 1.2 m long, and 1.0 m high) equipped with a stainless-steel self-feeder, a nipple water drinker, and iron mesh floor. All pigs were exposed to natural high AT conditions. The AT and relative humidity inside each room were recorded during the study with the aid of a Higrothermograph (Thermotracker HIGRO; iButtonLink LLC, Whitewater, WI, USA) set to record those values every hour. Pigs from each pair were randomly assigned to two dietary treatments (six replicates per treatment; Table 1): 1) high protein, 21.7% CP diet (HShp) and 2) low protein, 13.5% CP diet supplemented with crystalline AA (Lys, Thr, Met, Trp, His, Ile, Leu, Phe, and Val; HSaa). The HShp diet was formulated to meet NRC (2012) requirements but contained only protein-bound AA from wheat and soybean meal (SBM). Except for Lys and Met, the HShp diet contained an excess level of other eight indispensable AA. The HSaa diet was formulated to contain the same standardized ileal digestible (SID) Lys as for HShp diet and balancing other AA on the basis of the ideal protein concept by adding the first nine limiting AA in free form (except Arg which was already above requirement) and reducing inclusion of SBM to 4%. The dietary ingredients were analyzed for AA content (method 982.30; AOAC, 2006). The dietary SID contents of AA (Table 2) were calculated using the analyzed AA content and the SID coefficients for wheat and SBM reported by Stein et al. (2001). The feed intake of all pigs was restricted to approx. 95% of their ad libitum intake recorded during the previous week. Because we were interested in analyzing solely the effect of the form in which AA were supplied, free vs. protein-bound, all pigs were fed the same amount of feed without the confounding effect of differential feed intake. Hence meals were offered two times a day, at 0700 and 1900 hours. All pigs were trained to consume their daily meals within 30 min or less during 7 d before to the start of the trial. Purified water was available to all pigs. The average BW of the pigs at the end of the 21-d study was 58.5 ± 1.4 kg. Table 1. Ingredient composition of the experimental diets (as-fed basis) Ingredient, % . High protein (HShp) . Low protein + free AA (HSaa) . Wheat 65.10 91.62 Soybean meal, 48% 30.30 4.00 Canola oil 2.00 — l-Lys · HCl — 0.78 l-Thr — 0.26 dl-Met — 0.13 l-Trp — 0.05 l-Phe — 0.03 l-Leu — 0.13 l-Ile — 0.06 l-Val — 0.11 l-His — 0.03 Calcium carbonate 1.40 1.40 Dicalcium phosphate 0.65 0.65 Iodized salt 0.35 0.35 Vitamin and mineral premix1 0.40 0.40 Calculated SID AA content, %  NE, MJ/kg2 10.10 10.21  SID Lys 0.98 0.98  SID Met 0.28 0.28  SID Thr 0.68 0.59  SID Trp 0.25 0.18  SID Val 0.88 0.64 Ingredient, % . High protein (HShp) . Low protein + free AA (HSaa) . Wheat 65.10 91.62 Soybean meal, 48% 30.30 4.00 Canola oil 2.00 — l-Lys · HCl — 0.78 l-Thr — 0.26 dl-Met — 0.13 l-Trp — 0.05 l-Phe — 0.03 l-Leu — 0.13 l-Ile — 0.06 l-Val — 0.11 l-His — 0.03 Calcium carbonate 1.40 1.40 Dicalcium phosphate 0.65 0.65 Iodized salt 0.35 0.35 Vitamin and mineral premix1 0.40 0.40 Calculated SID AA content, %  NE, MJ/kg2 10.10 10.21  SID Lys 0.98 0.98  SID Met 0.28 0.28  SID Thr 0.68 0.59  SID Trp 0.25 0.18  SID Val 0.88 0.64 1Supplied per kg of diet: vitamin A, 4,800 IU; vitamin D3, 800 IU; vitamin E, 4.8 IU; vitamin K3, 1.6 mg; riboflavin, 4 mg; D-pantothenic acid, 7.2 mg; niacin, 16 mg; vitamin B12, 12.8 mg; Zn, 64 mg; Fe, 64 mg; Cu, 4 mg; Mn, 4 mg; I, 0.36 mg; Se, 0.13 mg. The premix was supplied by Nutrionix, S.A., Hermosillo, México. 2NE, MJ/kg: 10.21; 9.88. Open in new tab Table 1. Ingredient composition of the experimental diets (as-fed basis) Ingredient, % . High protein (HShp) . Low protein + free AA (HSaa) . Wheat 65.10 91.62 Soybean meal, 48% 30.30 4.00 Canola oil 2.00 — l-Lys · HCl — 0.78 l-Thr — 0.26 dl-Met — 0.13 l-Trp — 0.05 l-Phe — 0.03 l-Leu — 0.13 l-Ile — 0.06 l-Val — 0.11 l-His — 0.03 Calcium carbonate 1.40 1.40 Dicalcium phosphate 0.65 0.65 Iodized salt 0.35 0.35 Vitamin and mineral premix1 0.40 0.40 Calculated SID AA content, %  NE, MJ/kg2 10.10 10.21  SID Lys 0.98 0.98  SID Met 0.28 0.28  SID Thr 0.68 0.59  SID Trp 0.25 0.18  SID Val 0.88 0.64 Ingredient, % . High protein (HShp) . Low protein + free AA (HSaa) . Wheat 65.10 91.62 Soybean meal, 48% 30.30 4.00 Canola oil 2.00 — l-Lys · HCl — 0.78 l-Thr — 0.26 dl-Met — 0.13 l-Trp — 0.05 l-Phe — 0.03 l-Leu — 0.13 l-Ile — 0.06 l-Val — 0.11 l-His — 0.03 Calcium carbonate 1.40 1.40 Dicalcium phosphate 0.65 0.65 Iodized salt 0.35 0.35 Vitamin and mineral premix1 0.40 0.40 Calculated SID AA content, %  NE, MJ/kg2 10.10 10.21  SID Lys 0.98 0.98  SID Met 0.28 0.28  SID Thr 0.68 0.59  SID Trp 0.25 0.18  SID Val 0.88 0.64 1Supplied per kg of diet: vitamin A, 4,800 IU; vitamin D3, 800 IU; vitamin E, 4.8 IU; vitamin K3, 1.6 mg; riboflavin, 4 mg; D-pantothenic acid, 7.2 mg; niacin, 16 mg; vitamin B12, 12.8 mg; Zn, 64 mg; Fe, 64 mg; Cu, 4 mg; Mn, 4 mg; I, 0.36 mg; Se, 0.13 mg. The premix was supplied by Nutrionix, S.A., Hermosillo, México. 2NE, MJ/kg: 10.21; 9.88. Open in new tab Table 2. Analyzed and (calculated) AA composition of the experimental diets (as-fed basis) Amino acid, % . High protein . Low protein + free AA . Arg 1.50 (1.51) 0.80 (0.76) His 0.60 (0.61) 0.36 (0.38) Ile 1.00 (0.98) 0.61 (0.59) Leu 1.77 (1.75) 1.04 (1.14) Lys 1.13 (1.16) 1.09 (1.07) Met 0.35 (0.33) 0.30 (0.33) Phe 1.18 (1.16) 0.70 (0.70) Thr 0.83 (0.82) 0.62 (0.66) Val 1.11 (1.09) 0.70 (0.74) Amino acid, % . High protein . Low protein + free AA . Arg 1.50 (1.51) 0.80 (0.76) His 0.60 (0.61) 0.36 (0.38) Ile 1.00 (0.98) 0.61 (0.59) Leu 1.77 (1.75) 1.04 (1.14) Lys 1.13 (1.16) 1.09 (1.07) Met 0.35 (0.33) 0.30 (0.33) Phe 1.18 (1.16) 0.70 (0.70) Thr 0.83 (0.82) 0.62 (0.66) Val 1.11 (1.09) 0.70 (0.74) Open in new tab Table 2. Analyzed and (calculated) AA composition of the experimental diets (as-fed basis) Amino acid, % . High protein . Low protein + free AA . Arg 1.50 (1.51) 0.80 (0.76) His 0.60 (0.61) 0.36 (0.38) Ile 1.00 (0.98) 0.61 (0.59) Leu 1.77 (1.75) 1.04 (1.14) Lys 1.13 (1.16) 1.09 (1.07) Met 0.35 (0.33) 0.30 (0.33) Phe 1.18 (1.16) 0.70 (0.70) Thr 0.83 (0.82) 0.62 (0.66) Val 1.11 (1.09) 0.70 (0.74) Amino acid, % . High protein . Low protein + free AA . Arg 1.50 (1.51) 0.80 (0.76) His 0.60 (0.61) 0.36 (0.38) Ile 1.00 (0.98) 0.61 (0.59) Leu 1.77 (1.75) 1.04 (1.14) Lys 1.13 (1.16) 1.09 (1.07) Met 0.35 (0.33) 0.30 (0.33) Phe 1.18 (1.16) 0.70 (0.70) Thr 0.83 (0.82) 0.62 (0.66) Val 1.11 (1.09) 0.70 (0.74) Open in new tab Collection of blood and tissue samples Blood samples (approximately 7 mL) were collected by venipuncture of the jugular vein from all pigs to analyze the SC of free AA and some AA metabolites during both the absorptive and postabsorptive phases. On day 18 of the study, blood sampling was performed during the postabsorptive phase at 1700 hours, 2.0 h before the evening meal. On days 15 and 16 of the study, blood was collected during the absorptive phase at 2030 hours (1.5 h after the evening meal) and 2130 hours (2.5 h after the evening meal), respectively. This blood collection protocol was designed to minimize the additional stress pigs might experience. Immediately after collection, the blood samples were centrifuged at 1,500 × g, 4 °C for 10 min to separate serum from blood cells. Following, serum samples were freeze-dried and stored at −20 °C until analysis. On day 21, all pigs were euthanized by electrical stunning and exsanguination at about 2.5 h after the morning meal. The carcasses were immediately eviscerated. Mucosal samples scratched from jejunum and ileum (approximately 0.5 g) were collected into 2-mL microtubes and immediately frozen in liquid N, and stored at −82 °C until analysis for gene expression of AA transporters. Additionally, 5-cm sections from each segment of the small intestine of all pigs were collected and washed with physiological saline as described by Liu et al. (2009) for the determination of gut histomorphology. The total collection process took no longer than 8 min. Reverse transcription and quantitative PCR Approximately, 2 µg of total RNA was treated with 1 U of DNase I (1 U/µL; Thermo Scientific, Inc., Carlsbad, CA, USA) and 6 µL of 5× reverse transcription buffer in a 30-µL reaction completed with nuclease-free water (Thermo Scientific, Inc., Carlsbad, CA, USA); the reaction was carried out for 15 min at room temperature and another 15 min at 70 °C to stop the reaction. The reverse transcription was initiated with DNase-treated RNA samples, adding 1 µL of random primers (150 ng/µL, Invitrogen Corp., Carlsbad, CA, USA) and 1 µL of deoxynucleotide triphosphates solution (10 µM each); the reaction was incubated for 5 min at room temperature and then chilled on ice for 1 min. Following, 3 µL of nuclease-free water, 1 µL of ribonuclease inhibitor (40 U/µL; RiboLock, Thermo Scientific, Inc., Carlsbad, CA, USA), and 2 µL of 5× reverse transcription buffer were added to the reaction and incubated at 42 °C for 2 min to stabilize it before adding 1 µL of reverse transcriptase enzyme (200 U/µL; RevertAid H Minus, Thermo Scientific, Inc., Carlsbad, CA, USA). The reverse transcription reaction was incubated at 42 °C for 50 min, followed by 15 min at 70 °C, and chilled on ice to stop it. cDNA samples were quantified spectrophotometrically and diluted into a final concentration of 50 ng/µL. Specific primers for AA transporters and the 18S rRNA were designed according to their published sequences at the GenBank as follows. B0AT1 (SLC6A19): Sus scrofa (GenBank DQ231579), Forward, 8–28, 5′-TCTGTCCACAACAACTGCGAG-3′, Reverse, 212–193, 5′-CAGCGAAGTTCTCCTGCGTC-3′, 205 bp amplicon size. y+ LAT1 (SLC7A7) S. scrofa (GenBank NM_001110421), Forward, 664–683, 5′-TCAAGTGGGGAACCCTGGTA-3′, Reverse, 922–903, 5′-ATGGAGAGGGGCAGATTCCT-3′, 259 bp amplicon size. 18S rRNA S. scrofa (GenBank: AY265350), Forward, 229–248, 5′-ATCCGAGGGCCTCACTAAAC-3′, Reverse, 530–511, 5′-TAGAGGGACAAGTGGCGTTC-3′, 302 bp amplicon size. The housekeeping 18S rRNA was used as an endogenous control to normalize variations in mRNA. Before starting, end point polymerize chain reaction (PCR) was carried out to standardize amplification conditions for each pair of primers and in order to confirm the specificity of the PCR products related to its mRNA. A sample of every PCR product was sequenced at Genewiz (South Plainfield, NJ, USA). The sequencing results revealed that the products for y+ LAT1, B0AT1, and 18S rRNA have 100% homology with their corresponding expected sequences acquired from the virtual template sequences reported in the GenBank. The expression of genes (relative mRNA abundance) coding for y+ LAT1 and B0AT1 were estimated by quantitative PCR (qPCR) assays, using the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific, Inc., Carlsbad, CA, USA) into a CFX96-RealTime System (Bio-Rad, Herefordshire, England), and results were analyzed with the software CFX Manager 3.0 (Bio-Rad). The reactions for qPCR contained 50 ng of cDNA, 0.5 µM of each specific primer, 12.5 µL of 2× SYBR green/ROX qPCR Master Mix, and nuclease-free water to complete a final volume of 25 µL. The PCR conditions used for the amplification and quantification were an initial denaturing stage (95 °C for 1 min) followed by 45 cycles of amplification (denaturing at 95 °C for 15 s, annealing at 56 °C for 15 s, and extension at 72 °C for 30 s) and a melting curve program (60 to 90 °C). Fluorescence was measured at the end of every cycle and every 0.5 °C during the melting program. Three-replicate negative controls were used: qPCR reactions without DNA template, qPCR reactions with DNA template but no SYBR Mix, and qPCR reactions with DNA template but no primers. The melting curve of each specific qPCR product was analyzed to make sure that no primer dimers or nonspecific DNA products were quantitated. Results of quantitation of mRNA expression were analyzed according to the comparative Ct method, expressed as 2-ΔΔCt (Livak and Schmittgen, 2001) and normalized by 18S ribosomal RNA expression in each sample. Gut histomorphology Sections of duodenum, jejunum, and ileum were fixed in 10% neutral buffered formalin for paraffin embedding. Formalin-fixed samples of jejunum and ileum were stained with hematoxylin and eosin (Driscoll and Ryan, 1978). The mucosal structure was observed into an optic microscopy (HBO50 Primo Star, Zeiss, Mexico) using 40× magnification and microphotographs were obtained by a photographic camera (Canon, Tokyo, Japan). Villus height and crypt depth of at least 10 well-oriented villi were measured and analyzed using the software Image J2 (Curtis et al., 2017). Analyses of AA in the diets and free AA in serum Analyses of AA, except for tryptophan, in feed ingredients and diets were performed by HPLC with post-column ninhydrin derivatization and the use of a fluorescence detector, after acid hydrolysis with 6 N HCl (method 982.30E; AOAC, 2006). The SC of all free AAs was determined (method 982.30E; AOAC, 2006) by ion-exchange chromatography using a Biochrom 20 AA analyzer column (Biochrom Ltd., Cambridge, UK) with lithium buffers. The AA in serum were determined after dissolving the freeze-dried serum samples and precipitating proteins with sulfosalicylic acid and centrifugation (30 min at 10,000 turns/min; temperature 20 to 25 °C). The AA were quantified using the internal standard norleucine by measuring the absorption of reaction products with ninhydrin at 570 and 440 nm. Statistical analyses Analyses of performance, intestinal morphology, gene expression, and SC of AA data were performed by independent-sample t-tests according to a randomized complete block design. The model considered the fixed effect of dietary treatment and the individual pig was considered as the experimental unit. Analyses of the SC of AA data at the preprandial and postprandial times were performed according to a repeated measures design, where blood sampling was repeated within the pig. For each treatment, three contrasts were constructed to evaluate the effect of blood sampling time on the SC of AA (C1, 2.0 h preprandial vs. 1.5 h postprandial; C2, 2.0 h preprandial vs. 2.5 h postprandial; C3, 1.5 h postprandial vs. 2.5 h postprandial). Probability levels of P ≤ 0.05 and 0.05 < P ≤ 0.10 were defined as significant differences and tendencies, respectively. Results Pigs remained healthy during the experiment despite their exposure to severe HS conditions. The AT recorded daily inside the room varied substantially within the same day; the lowest and highest AT were 24.5 to 42.6 °C, respectively (Figure 1). On average, the lowest AT was recorded from 0530 to 0630 hours. The AT above 40.0 °C was recorded between 1200 and 1830 hours. The performance results are presented in Table 3. The ADG did not differ among the HShp and HSaa pigs. Because all pigs, by design, were offered the same amount of feed every day, the daily intake of feed, Lys, Thr, and Met did not differ among treatments. Therefore, there was no effect of dietary treatment on G:F. Figure 1. Open in new tabDownload slide Hourly variations in ambient temperature recorded during the 21-d period of the experiment (each data point is the average of 21 recordings; SD = 1.51). Figure 1. Open in new tabDownload slide Hourly variations in ambient temperature recorded during the 21-d period of the experiment (each data point is the average of 21 recordings; SD = 1.51). Table 3. Performance of HS pigs fed a high protein (HShp) or a low protein diet supplemented with free AA (HSaa) . Treatment . . . Item . HShp . HSaa . SEM . P-value . Initial body weight, kg 47.3 47.5 Final body weight, kg 58.6 58.4 1.0 0.887 Daily weight gain, kg/d 0.540 0.520 0.025 0.589 Daily intake  Feed, kg/d 1.185 1.192 0.04 0.274  Lys, g/d 11.62 11.68 0.04 0.274  Thr, g/d 8.06 8.03 0.03 0.274  Met, g/d 3.32 3.34 0.01 0.274 G:F 0.456 0.436 0.02 0.761 . Treatment . . . Item . HShp . HSaa . SEM . P-value . Initial body weight, kg 47.3 47.5 Final body weight, kg 58.6 58.4 1.0 0.887 Daily weight gain, kg/d 0.540 0.520 0.025 0.589 Daily intake  Feed, kg/d 1.185 1.192 0.04 0.274  Lys, g/d 11.62 11.68 0.04 0.274  Thr, g/d 8.06 8.03 0.03 0.274  Met, g/d 3.32 3.34 0.01 0.274 G:F 0.456 0.436 0.02 0.761 Open in new tab Table 3. Performance of HS pigs fed a high protein (HShp) or a low protein diet supplemented with free AA (HSaa) . Treatment . . . Item . HShp . HSaa . SEM . P-value . Initial body weight, kg 47.3 47.5 Final body weight, kg 58.6 58.4 1.0 0.887 Daily weight gain, kg/d 0.540 0.520 0.025 0.589 Daily intake  Feed, kg/d 1.185 1.192 0.04 0.274  Lys, g/d 11.62 11.68 0.04 0.274  Thr, g/d 8.06 8.03 0.03 0.274  Met, g/d 3.32 3.34 0.01 0.274 G:F 0.456 0.436 0.02 0.761 . Treatment . . . Item . HShp . HSaa . SEM . P-value . Initial body weight, kg 47.3 47.5 Final body weight, kg 58.6 58.4 1.0 0.887 Daily weight gain, kg/d 0.540 0.520 0.025 0.589 Daily intake  Feed, kg/d 1.185 1.192 0.04 0.274  Lys, g/d 11.62 11.68 0.04 0.274  Thr, g/d 8.06 8.03 0.03 0.274  Met, g/d 3.32 3.34 0.01 0.274 G:F 0.456 0.436 0.02 0.761 Open in new tab The results of the intestinal morphology are presented in Figure 2. The villi height in duodenum and jejunum tended to be larger in pigs fed the HSaa diet (P ≤ 0.10) but no difference was observed in the ileum. The crypt depth did not differ among treatments in any of the intestinal segments. The villi height:crypt depth ratio in duodenum and jejunum was greater in pigs fed the HSaa diet (P < 0.05), but no difference was observed in ileum between the HShp and the HSaa diets. As shown in Table 4, the abundance of the mRNA expression for the cationic AA transporter y+L tended to be lower in the jejunum of pigs fed the HSaa diet (P = 0.068) but the neutral AA transporter B0 abundance was not affected. In Ileum, the mRNA abundance of the AA transporter B0 was lower in pigs fed the HSaa diet (P = 0.029), but the abundance of the y+L transporter did not differ among HSaa and HShp pigs. Figure 2. Open in new tabDownload slide Histomorphology of duodenum, jejunum, and ileum of HS pigs fed a high protein diet or a low protein diet supplemented with free AA. Figure 2. Open in new tabDownload slide Histomorphology of duodenum, jejunum, and ileum of HS pigs fed a high protein diet or a low protein diet supplemented with free AA. Table 4. Relative expression of mRNA coding for the AA transporters y+L and B0 in the jejunum and ileum of HS pigs fed a high protein diet or a low protein diet supplemented with free AA (arbitrary units) . . Treatment . . . Amino acid transporter . Intestinal segment . HShp . HSaa . SEM . P-value . y+L Jejunum 1.00 0.250 0.167 0.072 Ileum 1.00 0.833 0.054 0.638 B0 Jejunum 1.00 0.582 0.092 0.377 Ileum 1.00 0.327 0.071 0.036 . . Treatment . . . Amino acid transporter . Intestinal segment . HShp . HSaa . SEM . P-value . y+L Jejunum 1.00 0.250 0.167 0.072 Ileum 1.00 0.833 0.054 0.638 B0 Jejunum 1.00 0.582 0.092 0.377 Ileum 1.00 0.327 0.071 0.036 Open in new tab Table 4. Relative expression of mRNA coding for the AA transporters y+L and B0 in the jejunum and ileum of HS pigs fed a high protein diet or a low protein diet supplemented with free AA (arbitrary units) . . Treatment . . . Amino acid transporter . Intestinal segment . HShp . HSaa . SEM . P-value . y+L Jejunum 1.00 0.250 0.167 0.072 Ileum 1.00 0.833 0.054 0.638 B0 Jejunum 1.00 0.582 0.092 0.377 Ileum 1.00 0.327 0.071 0.036 . . Treatment . . . Amino acid transporter . Intestinal segment . HShp . HSaa . SEM . P-value . y+L Jejunum 1.00 0.250 0.167 0.072 Ileum 1.00 0.833 0.054 0.638 B0 Jejunum 1.00 0.582 0.092 0.377 Ileum 1.00 0.327 0.071 0.036 Open in new tab The SC (mg/dL) of indispensable AA in pigs fed the HShp or the HSaa diet at 1700 hours (10 h after morning meal and 2.5 h before the evening meal) and at 2030 and 2130 hours (1.5 and 2.5 h postprandial, respectively) is presented in Table 5. At 1700 hours, the SC of Arg, His, Ile, Leu, Thr, Trp, and Val was lower (P ≤ 0.050) and that of Phe tended to be lower (P = 0.063) in pigs fed the HSaa diet, but no difference was observed in the SC of Lys and Met. At 2030 hours, the SC of Lys, Met, and Thr was higher in the HSaa pigs (P ≤ 0.027) but the SC of Arg, His, Ile, Leu, Phe, Trp, and Val did not differ between treatments. At 2130 hours, the SC of Arg, His, Ile, Phe, and Trp was lower (P ≤ 0.018) that of Met was higher (P ≤ 0.050), and Lys tended to be higher (P = 0.092) in HSaa pigs; the SC of Leu, Thr, and Val did not differ between dietary treatments. Table 5. Serum concentrations (mg/dL) of indispensable AA analyzed at 2.0 h preprandial (pre—1700 hours), and at 1.5 h (post—2030 hours) and 2.5 h (post—2130 hours) postprandial in HS pigs fed a high protein diet or a low protein diet supplemented with free AA Feeding time . Amino acid . HShp diet . HSaa diet . SEM . P-value . 1700 hours (postabsorptive) Arg 28.58 21.30 2.82 0.050 His 11.88 3.89 0.76 <0.001 Ile 17.79 8.86 1.37 0.001 Leu 24.86 16.79 2.47 0.019 Lys 11.52 9.71 1.89 0.231 Met 2.94 8.82 0.81 0.416 Phe 13.88 10.52 1.57 0.063 Thr 20.62 15.24 2.21 0.036 Trp 10.98 5.93 0.94 0.005 Val 35.89 25.03 3.49 0.019 2030 hours (absorptive) Arg 32.82 36.7 5.86 0.719 His 10.91 7.95 1.16 0.127 Ile 17.79 16.41 1.99 0.801 Leu 26.77 34.05 2.8 0.100 Lys 18.99 48.02 6.28 0.017 Met 2.86 10.18 1.71 0.023 Phe 15.00 17.65 1.73 0.317 Thr 24.35 31.1 1.94 0.027 Trp 12.25 11.22 1.44 0.630 Val 38.08 43.04 3.06 0.342 2130 hours (absorptive) Arg 54.23 32.99 1.14 < 0.001 His 17.43 8.3 1.2 0.002 Ile 24.24 14.11 1.78 0.007 Leu 35.42 29.1 3.88 0.293 Lys 27.72 40.98 4.68 0.092 Met 4.42 10.16 1.23 0.016 Phe 22.25 16.69 0.92 0.005 Thr 33.33 28.62 2.89 0.294 Trp 15.11 9.98 1.13 0.018 Val 49.84 39.48 0.404 0.120 Feeding time . Amino acid . HShp diet . HSaa diet . SEM . P-value . 1700 hours (postabsorptive) Arg 28.58 21.30 2.82 0.050 His 11.88 3.89 0.76 <0.001 Ile 17.79 8.86 1.37 0.001 Leu 24.86 16.79 2.47 0.019 Lys 11.52 9.71 1.89 0.231 Met 2.94 8.82 0.81 0.416 Phe 13.88 10.52 1.57 0.063 Thr 20.62 15.24 2.21 0.036 Trp 10.98 5.93 0.94 0.005 Val 35.89 25.03 3.49 0.019 2030 hours (absorptive) Arg 32.82 36.7 5.86 0.719 His 10.91 7.95 1.16 0.127 Ile 17.79 16.41 1.99 0.801 Leu 26.77 34.05 2.8 0.100 Lys 18.99 48.02 6.28 0.017 Met 2.86 10.18 1.71 0.023 Phe 15.00 17.65 1.73 0.317 Thr 24.35 31.1 1.94 0.027 Trp 12.25 11.22 1.44 0.630 Val 38.08 43.04 3.06 0.342 2130 hours (absorptive) Arg 54.23 32.99 1.14 < 0.001 His 17.43 8.3 1.2 0.002 Ile 24.24 14.11 1.78 0.007 Leu 35.42 29.1 3.88 0.293 Lys 27.72 40.98 4.68 0.092 Met 4.42 10.16 1.23 0.016 Phe 22.25 16.69 0.92 0.005 Thr 33.33 28.62 2.89 0.294 Trp 15.11 9.98 1.13 0.018 Val 49.84 39.48 0.404 0.120 Open in new tab Table 5. Serum concentrations (mg/dL) of indispensable AA analyzed at 2.0 h preprandial (pre—1700 hours), and at 1.5 h (post—2030 hours) and 2.5 h (post—2130 hours) postprandial in HS pigs fed a high protein diet or a low protein diet supplemented with free AA Feeding time . Amino acid . HShp diet . HSaa diet . SEM . P-value . 1700 hours (postabsorptive) Arg 28.58 21.30 2.82 0.050 His 11.88 3.89 0.76 <0.001 Ile 17.79 8.86 1.37 0.001 Leu 24.86 16.79 2.47 0.019 Lys 11.52 9.71 1.89 0.231 Met 2.94 8.82 0.81 0.416 Phe 13.88 10.52 1.57 0.063 Thr 20.62 15.24 2.21 0.036 Trp 10.98 5.93 0.94 0.005 Val 35.89 25.03 3.49 0.019 2030 hours (absorptive) Arg 32.82 36.7 5.86 0.719 His 10.91 7.95 1.16 0.127 Ile 17.79 16.41 1.99 0.801 Leu 26.77 34.05 2.8 0.100 Lys 18.99 48.02 6.28 0.017 Met 2.86 10.18 1.71 0.023 Phe 15.00 17.65 1.73 0.317 Thr 24.35 31.1 1.94 0.027 Trp 12.25 11.22 1.44 0.630 Val 38.08 43.04 3.06 0.342 2130 hours (absorptive) Arg 54.23 32.99 1.14 < 0.001 His 17.43 8.3 1.2 0.002 Ile 24.24 14.11 1.78 0.007 Leu 35.42 29.1 3.88 0.293 Lys 27.72 40.98 4.68 0.092 Met 4.42 10.16 1.23 0.016 Phe 22.25 16.69 0.92 0.005 Thr 33.33 28.62 2.89 0.294 Trp 15.11 9.98 1.13 0.018 Val 49.84 39.48 0.404 0.120 Feeding time . Amino acid . HShp diet . HSaa diet . SEM . P-value . 1700 hours (postabsorptive) Arg 28.58 21.30 2.82 0.050 His 11.88 3.89 0.76 <0.001 Ile 17.79 8.86 1.37 0.001 Leu 24.86 16.79 2.47 0.019 Lys 11.52 9.71 1.89 0.231 Met 2.94 8.82 0.81 0.416 Phe 13.88 10.52 1.57 0.063 Thr 20.62 15.24 2.21 0.036 Trp 10.98 5.93 0.94 0.005 Val 35.89 25.03 3.49 0.019 2030 hours (absorptive) Arg 32.82 36.7 5.86 0.719 His 10.91 7.95 1.16 0.127 Ile 17.79 16.41 1.99 0.801 Leu 26.77 34.05 2.8 0.100 Lys 18.99 48.02 6.28 0.017 Met 2.86 10.18 1.71 0.023 Phe 15.00 17.65 1.73 0.317 Thr 24.35 31.1 1.94 0.027 Trp 12.25 11.22 1.44 0.630 Val 38.08 43.04 3.06 0.342 2130 hours (absorptive) Arg 54.23 32.99 1.14 < 0.001 His 17.43 8.3 1.2 0.002 Ile 24.24 14.11 1.78 0.007 Leu 35.42 29.1 3.88 0.293 Lys 27.72 40.98 4.68 0.092 Met 4.42 10.16 1.23 0.016 Phe 22.25 16.69 0.92 0.005 Thr 33.33 28.62 2.89 0.294 Trp 15.11 9.98 1.13 0.018 Val 49.84 39.48 0.404 0.120 Open in new tab The postprandial (2030 and 2130 hours) change (%) in the SC of indispensable AA relative to the preprandial SC (1700 hours) is shown in Figure 3. In pigs fed the HShp diet (panel A), the SC of Lys at 2030 hours (1.5 h postprandial) tended to be higher than the preprandial SC (P < 0.10) but the SC of the other indispensable AA did not differ among these sampling times. However, at 2130 hours (2.5 h postprandial), the SC of all indispensable AA was higher than the preprandial SC (P < 0.05). In pigs fed the HSaa diet (panel B), the SC of all indispensable AA at both 1.5 h and 2.5 h postprandial was higher than the preprandial SC (P < 0.05). When comparing the SC between postprandial times (panel C), no difference was observed in pigs fed the HSaa diet, but in pigs fed the HShp diet the SC at 2130 hours was higher than that at 2030 hours (P < 0.10). Figure 3. Open in new tabDownload slide Serum concentration difference (%) of indispensable AA between pre- and post-prandial times (1700 vs. 2030 or 2130 hours) in HSaa pigs (panel A) or HShp pigs (panel B), and between postprandial times (2030 vs. 2130 hours) in pigs fed the HShp or the HSaa diet (panel C). Figure 3. Open in new tabDownload slide Serum concentration difference (%) of indispensable AA between pre- and post-prandial times (1700 vs. 2030 or 2130 hours) in HSaa pigs (panel A) or HShp pigs (panel B), and between postprandial times (2030 vs. 2130 hours) in pigs fed the HShp or the HSaa diet (panel C). The SC (mg/dL) of dispensable AA in pigs fed the HShp or the HSaa diet at 1700 hours (2.5 h preprandial), 2030 hours, and 2130 hours (1.5 and 2.5 h postprandial, respectively) are presented in Table 6. At 1700 hours, the SC of Asn, Asp, and Tyr was lower in the HSaa pigs (P < 0.05); however, no difference in the SC of the other dispensable AA was observed between the HShp and HSaa pigs. At 2030 hours, serum Ala, Glu, Gln, and Gly were higher in HSaa pigs (P < 0.05). At 2130 hours, serum Ala and Glu were higher, and Asn and Tyr were lower in the HSaa pigs (P < 0.05). There was a tendency (P < 0.10) for Cys to be lower and for Gln to be higher in the HSaa pigs but no difference was observed in Asp, Gly, Pro, and Ser between the treatments. Table 6. Serum concentrations of dispensable AA analyzed at 2.0 h preprandial (pre—1700 hours), and at 1.5 h (post—2030 hours) and 2.5 h (post—2130 hours) postprandial in HS pigs fed a high protein diet or a low protein diet supplemented with free AA Feeding time . Amino acid . HShp diet . HSaa diet . SEM . P-value . 1700 hours (postabsorptive) Ala 38.24 52.71 9.01 0.302 Asn 9.08 3.36 0.79 0.002 Asp 5.83 2.77 0.60 0.011 Cys 0.46 0.04 0.16 0.130 Glu 21.91 22.89 4.18 0.874 Gln 55.50 65.46 12.41 0.591 Gly 57.63 60.80 11.63 0.854 Pro 39.56 28.13 4.60 0.129 Ser 18.36 14.95 3.60 0.528 Tyr 19.68 6.64 1.48 <0.001 2030 hours (absorptive) Ala 49.08 97.84 10.11 0.014 Asn 10.96 8.95 1.71 0.437 Asp 5.08 5.55 0.57 0.581 Cys 0.69 0.47 0.28 0.603 Glu 17.23 45.35 4.88 0.007 Gln 60.14 125.21 11.05 0.006 Gly 54.47 89.75 7.69 0.018 Pro 50.91 65.45 7.65 0.227 Ser 22.28 27.58 3.89 0.311 Tyr 19.86 14.36 2.60 0.186 2130 hours (absorptive) Ala 55.33 92.42 9.50 0.033 Asn 20.26 7.84 1.75 0.002 Asp 5.27 4.80 0.75 0.669 Cys 1.30 0.32 0.34 0.086 Glu 19.45 38.17 4.03 0.017 Gln 85.25 118.12 11.68 0.094 Gly 74.55 82.70 12.24 0.654 Pro 64.15 66.77 9.93 0.858 Ser 27.90 24.90 6.04 0.738 Tyr 28.45 12.17 1.37 <0.001 Feeding time . Amino acid . HShp diet . HSaa diet . SEM . P-value . 1700 hours (postabsorptive) Ala 38.24 52.71 9.01 0.302 Asn 9.08 3.36 0.79 0.002 Asp 5.83 2.77 0.60 0.011 Cys 0.46 0.04 0.16 0.130 Glu 21.91 22.89 4.18 0.874 Gln 55.50 65.46 12.41 0.591 Gly 57.63 60.80 11.63 0.854 Pro 39.56 28.13 4.60 0.129 Ser 18.36 14.95 3.60 0.528 Tyr 19.68 6.64 1.48 <0.001 2030 hours (absorptive) Ala 49.08 97.84 10.11 0.014 Asn 10.96 8.95 1.71 0.437 Asp 5.08 5.55 0.57 0.581 Cys 0.69 0.47 0.28 0.603 Glu 17.23 45.35 4.88 0.007 Gln 60.14 125.21 11.05 0.006 Gly 54.47 89.75 7.69 0.018 Pro 50.91 65.45 7.65 0.227 Ser 22.28 27.58 3.89 0.311 Tyr 19.86 14.36 2.60 0.186 2130 hours (absorptive) Ala 55.33 92.42 9.50 0.033 Asn 20.26 7.84 1.75 0.002 Asp 5.27 4.80 0.75 0.669 Cys 1.30 0.32 0.34 0.086 Glu 19.45 38.17 4.03 0.017 Gln 85.25 118.12 11.68 0.094 Gly 74.55 82.70 12.24 0.654 Pro 64.15 66.77 9.93 0.858 Ser 27.90 24.90 6.04 0.738 Tyr 28.45 12.17 1.37 <0.001 Open in new tab Table 6. Serum concentrations of dispensable AA analyzed at 2.0 h preprandial (pre—1700 hours), and at 1.5 h (post—2030 hours) and 2.5 h (post—2130 hours) postprandial in HS pigs fed a high protein diet or a low protein diet supplemented with free AA Feeding time . Amino acid . HShp diet . HSaa diet . SEM . P-value . 1700 hours (postabsorptive) Ala 38.24 52.71 9.01 0.302 Asn 9.08 3.36 0.79 0.002 Asp 5.83 2.77 0.60 0.011 Cys 0.46 0.04 0.16 0.130 Glu 21.91 22.89 4.18 0.874 Gln 55.50 65.46 12.41 0.591 Gly 57.63 60.80 11.63 0.854 Pro 39.56 28.13 4.60 0.129 Ser 18.36 14.95 3.60 0.528 Tyr 19.68 6.64 1.48 <0.001 2030 hours (absorptive) Ala 49.08 97.84 10.11 0.014 Asn 10.96 8.95 1.71 0.437 Asp 5.08 5.55 0.57 0.581 Cys 0.69 0.47 0.28 0.603 Glu 17.23 45.35 4.88 0.007 Gln 60.14 125.21 11.05 0.006 Gly 54.47 89.75 7.69 0.018 Pro 50.91 65.45 7.65 0.227 Ser 22.28 27.58 3.89 0.311 Tyr 19.86 14.36 2.60 0.186 2130 hours (absorptive) Ala 55.33 92.42 9.50 0.033 Asn 20.26 7.84 1.75 0.002 Asp 5.27 4.80 0.75 0.669 Cys 1.30 0.32 0.34 0.086 Glu 19.45 38.17 4.03 0.017 Gln 85.25 118.12 11.68 0.094 Gly 74.55 82.70 12.24 0.654 Pro 64.15 66.77 9.93 0.858 Ser 27.90 24.90 6.04 0.738 Tyr 28.45 12.17 1.37 <0.001 Feeding time . Amino acid . HShp diet . HSaa diet . SEM . P-value . 1700 hours (postabsorptive) Ala 38.24 52.71 9.01 0.302 Asn 9.08 3.36 0.79 0.002 Asp 5.83 2.77 0.60 0.011 Cys 0.46 0.04 0.16 0.130 Glu 21.91 22.89 4.18 0.874 Gln 55.50 65.46 12.41 0.591 Gly 57.63 60.80 11.63 0.854 Pro 39.56 28.13 4.60 0.129 Ser 18.36 14.95 3.60 0.528 Tyr 19.68 6.64 1.48 <0.001 2030 hours (absorptive) Ala 49.08 97.84 10.11 0.014 Asn 10.96 8.95 1.71 0.437 Asp 5.08 5.55 0.57 0.581 Cys 0.69 0.47 0.28 0.603 Glu 17.23 45.35 4.88 0.007 Gln 60.14 125.21 11.05 0.006 Gly 54.47 89.75 7.69 0.018 Pro 50.91 65.45 7.65 0.227 Ser 22.28 27.58 3.89 0.311 Tyr 19.86 14.36 2.60 0.186 2130 hours (absorptive) Ala 55.33 92.42 9.50 0.033 Asn 20.26 7.84 1.75 0.002 Asp 5.27 4.80 0.75 0.669 Cys 1.30 0.32 0.34 0.086 Glu 19.45 38.17 4.03 0.017 Gln 85.25 118.12 11.68 0.094 Gly 74.55 82.70 12.24 0.654 Pro 64.15 66.77 9.93 0.858 Ser 27.90 24.90 6.04 0.738 Tyr 28.45 12.17 1.37 <0.001 Open in new tab The SC of AA metabolites at 1700 hours (2.5 h preprandial) and at 2030 and 2130 hours (1.5 and 2.5 h postprandial, respectively) in pigs fed either the HShp or the HSaa diet is presented in Table 7. At 1700 hours, the SC of 3-methyl-His, phospho-Ser, ornithine, carnosine, Tau, and urea was lower (P < 0.05), and OH-Lys tended to be lower (P = 0.10) in pigs fed the HSaa diet. At 2030 hours, the SC of 3-methyl-His, phospho-Ser, and urea was also lower, but citrulline was higher (P < 0.05) and OH-Pro tended to be higher (P < 0.10) in pigs fed the HSaa diet. At 2130 hours, the postprandial SC of 3-methyl-His, ornithine, carnosine, and urea was lower (P < 0.05), and OH-Lys and phospho-Ser tended to be lower (P < 0.10) in pigs fed the HSaa diet. Table 7. Serum concentrations of AA metabolites analyzed at 2.0 h preprandial (pre—1700 hours), and at 1.5 (post—2030 hours) and 2.5 (post—2130 hours) h postprandial in HS pigs fed a high protein diet or a low protein diet supplemented with free AA Feeding time . AA . HShp diet . HSaa diet . SEM . P-value . Pre—1700 hours (postabsorptive) 3-Methyl-His 1.47 0.49 0.12 0.001 OH-Lys 0.71 0.27 0.16 0.100 OH-Pro 11.48 11.54 2.40 0.986 Phospho-Ser 3.17 1.61 0.28 0.007 Citrulline 10.38 9.03 1.79 0.613 Ornithine 12.16 7.04 1.45 0.047 Carnosine 4.86 1.7 0.37 <0.001 Sarcosine 1.78 1.26 0.36 0.336 Taurine 19.13 10.41 2.25 0.034 Urea 281.7 79.3 15.22 <0.001 Post—2030 hours (absorptive) 3-Methyl-His 1.43 0.82 0.15 0.028 OH-Lys 1.04 0.46 0.43 0.378 OH-Pro 12.96 18.5 1.86 0.076 Phospho-Ser 3.43 1.91 0.33 0.017 Citrulline 11.11 23.2 3.27 0.040 Ornithine 15.27 16.13 2.32 0.801 Carnosine 3.56 3.27 0.51 0.712 Sarcosine 2.38 1.82 0.43 0.392 Taurine 17.21 20.35 1.76 0.255 Urea 274.1 133.2 18.6 0.002 Post—2130 hours (absorptive) 3-Methyl-His 1.69 0.78 0.09 <0.001 OH-Lys 1.23 0.37 0.28 0.077 OH-Pro 14.14 16.33 2.12 0.492 Phospho-Ser 2.96 1.88 0.34 0.067 Citrulline 16.09 17.03 2.37 0.790 Ornithine 22.56 13.51 2.00 0.019 Carnosine 5.7 2.6 0.28 <0.001 Sarcosine 2.41 1.74 0.61 0.468 Taurine 19.19 17.43 1.91 0.538 Urea 320.5 140.5 25.9 0.003 Feeding time . AA . HShp diet . HSaa diet . SEM . P-value . Pre—1700 hours (postabsorptive) 3-Methyl-His 1.47 0.49 0.12 0.001 OH-Lys 0.71 0.27 0.16 0.100 OH-Pro 11.48 11.54 2.40 0.986 Phospho-Ser 3.17 1.61 0.28 0.007 Citrulline 10.38 9.03 1.79 0.613 Ornithine 12.16 7.04 1.45 0.047 Carnosine 4.86 1.7 0.37 <0.001 Sarcosine 1.78 1.26 0.36 0.336 Taurine 19.13 10.41 2.25 0.034 Urea 281.7 79.3 15.22 <0.001 Post—2030 hours (absorptive) 3-Methyl-His 1.43 0.82 0.15 0.028 OH-Lys 1.04 0.46 0.43 0.378 OH-Pro 12.96 18.5 1.86 0.076 Phospho-Ser 3.43 1.91 0.33 0.017 Citrulline 11.11 23.2 3.27 0.040 Ornithine 15.27 16.13 2.32 0.801 Carnosine 3.56 3.27 0.51 0.712 Sarcosine 2.38 1.82 0.43 0.392 Taurine 17.21 20.35 1.76 0.255 Urea 274.1 133.2 18.6 0.002 Post—2130 hours (absorptive) 3-Methyl-His 1.69 0.78 0.09 <0.001 OH-Lys 1.23 0.37 0.28 0.077 OH-Pro 14.14 16.33 2.12 0.492 Phospho-Ser 2.96 1.88 0.34 0.067 Citrulline 16.09 17.03 2.37 0.790 Ornithine 22.56 13.51 2.00 0.019 Carnosine 5.7 2.6 0.28 <0.001 Sarcosine 2.41 1.74 0.61 0.468 Taurine 19.19 17.43 1.91 0.538 Urea 320.5 140.5 25.9 0.003 Open in new tab Table 7. Serum concentrations of AA metabolites analyzed at 2.0 h preprandial (pre—1700 hours), and at 1.5 (post—2030 hours) and 2.5 (post—2130 hours) h postprandial in HS pigs fed a high protein diet or a low protein diet supplemented with free AA Feeding time . AA . HShp diet . HSaa diet . SEM . P-value . Pre—1700 hours (postabsorptive) 3-Methyl-His 1.47 0.49 0.12 0.001 OH-Lys 0.71 0.27 0.16 0.100 OH-Pro 11.48 11.54 2.40 0.986 Phospho-Ser 3.17 1.61 0.28 0.007 Citrulline 10.38 9.03 1.79 0.613 Ornithine 12.16 7.04 1.45 0.047 Carnosine 4.86 1.7 0.37 <0.001 Sarcosine 1.78 1.26 0.36 0.336 Taurine 19.13 10.41 2.25 0.034 Urea 281.7 79.3 15.22 <0.001 Post—2030 hours (absorptive) 3-Methyl-His 1.43 0.82 0.15 0.028 OH-Lys 1.04 0.46 0.43 0.378 OH-Pro 12.96 18.5 1.86 0.076 Phospho-Ser 3.43 1.91 0.33 0.017 Citrulline 11.11 23.2 3.27 0.040 Ornithine 15.27 16.13 2.32 0.801 Carnosine 3.56 3.27 0.51 0.712 Sarcosine 2.38 1.82 0.43 0.392 Taurine 17.21 20.35 1.76 0.255 Urea 274.1 133.2 18.6 0.002 Post—2130 hours (absorptive) 3-Methyl-His 1.69 0.78 0.09 <0.001 OH-Lys 1.23 0.37 0.28 0.077 OH-Pro 14.14 16.33 2.12 0.492 Phospho-Ser 2.96 1.88 0.34 0.067 Citrulline 16.09 17.03 2.37 0.790 Ornithine 22.56 13.51 2.00 0.019 Carnosine 5.7 2.6 0.28 <0.001 Sarcosine 2.41 1.74 0.61 0.468 Taurine 19.19 17.43 1.91 0.538 Urea 320.5 140.5 25.9 0.003 Feeding time . AA . HShp diet . HSaa diet . SEM . P-value . Pre—1700 hours (postabsorptive) 3-Methyl-His 1.47 0.49 0.12 0.001 OH-Lys 0.71 0.27 0.16 0.100 OH-Pro 11.48 11.54 2.40 0.986 Phospho-Ser 3.17 1.61 0.28 0.007 Citrulline 10.38 9.03 1.79 0.613 Ornithine 12.16 7.04 1.45 0.047 Carnosine 4.86 1.7 0.37 <0.001 Sarcosine 1.78 1.26 0.36 0.336 Taurine 19.13 10.41 2.25 0.034 Urea 281.7 79.3 15.22 <0.001 Post—2030 hours (absorptive) 3-Methyl-His 1.43 0.82 0.15 0.028 OH-Lys 1.04 0.46 0.43 0.378 OH-Pro 12.96 18.5 1.86 0.076 Phospho-Ser 3.43 1.91 0.33 0.017 Citrulline 11.11 23.2 3.27 0.040 Ornithine 15.27 16.13 2.32 0.801 Carnosine 3.56 3.27 0.51 0.712 Sarcosine 2.38 1.82 0.43 0.392 Taurine 17.21 20.35 1.76 0.255 Urea 274.1 133.2 18.6 0.002 Post—2130 hours (absorptive) 3-Methyl-His 1.69 0.78 0.09 <0.001 OH-Lys 1.23 0.37 0.28 0.077 OH-Pro 14.14 16.33 2.12 0.492 Phospho-Ser 2.96 1.88 0.34 0.067 Citrulline 16.09 17.03 2.37 0.790 Ornithine 22.56 13.51 2.00 0.019 Carnosine 5.7 2.6 0.28 <0.001 Sarcosine 2.41 1.74 0.61 0.468 Taurine 19.19 17.43 1.91 0.538 Urea 320.5 140.5 25.9 0.003 Open in new tab Discussion The optimum AT or thermal neutral zone of growing pigs ranges from 15 to 25 °C (Federation of Animal Science Societies, 2010). Consequently, the exposure to more than 25 °C is expected to create HS condition. Indeed, pigs exposed to 33 °C or above show marked signs of HS ( Collin et al., 2001; Renaudeau et al., 2011). As pigs were exposed to 25 °C or higher all the time, and to 33 °C or above (up to 42.6 °C) for around 12 h every day, all pigs were exposed to moderate or severe HS conditions in the current experiment. On the other hand, differences in absorption between dietary protein-bound AA and free AA have been reported in thermal neutral pigs (Yen et al., 2004; Morales et al., 2015), but information in HS growing pigs is limited. Pigs can maintain their body temperature relatively constant when exposed to AT within the thermal neutral range; however, above this, optimal range pigs reduce feed intake and physical activity and increase the respiration rate as an effort to prevent excess body heat (Huynh et al., 2005; Pearce et al., 2013; Cervantes et al., 2017). When AT reaches high critical levels, however, these modifications are not sufficient to maintain body temperature unchanged. Pigs exposed constantly to 35 °C (Pearce et al., 2013) or a range of 26 to 40 °C (Liu et al., 2009; Yu et al., 2010) increase their rectal temperature by 1.5 to 2.0 °C. We previously reported an increase in intestinal temperature up to 2.6 °C of pigs exposed to natural AT ranging from 26 to 40 °C (Morales et al., 2016; Cervantes et al., 2017). Because pigs in the present experiment were alike and facilities were the same as those used before (Morales et al., 2016; Cervantes et al., 2017), we believe that the intestinal temperature might have also increased up to 2 °C. On the other hand, the heat produced during the ingestion–digestion–metabolism processes seems to be affected by the composition of the diet. Pesta and Samuel (2014) reported that heat production is highest for protein (≈15% to 30% above the basal metabolic rate), as compared with carbohydrates (≈5% to 10%) and fat (≈ 0.3%). The increased metabolic rate in animals consuming high protein diets may indicate a higher heat production, which might be translated into higher body temperature. In fact, body temperature slightly increased when thermal neutral or HS pigs were fed a 21.6% CP diet as compared with their peers fed a 10.8% CP diet (Morales et al., 2018, 2019). Physiological and histological alterations in the small intestine occur as pigs transfer internal heat to the body surface to maintain unaltered the body temperature (Huynh et al., 2005) but reduce the blood flow to internal organs (Ooue et al., 2007). This blood flow redirection lowers the supply of oxygen and nutrients to the gastrointestinal tract that may damage the intestinal epithelia (Liu et al., 2009). Indeed, the exposure of pigs to HS reduced the small intestine villi height (Yu et al., 2010; Pearce et al., 2014). We also observed a significant reduction in the small intestine villi height (unpublished data) of growing pigs exposed to HS. In the present experiment, the increased villi height and villi height:crypt depth ratio observed in pigs fed the HSaa diet indicate that substituting protein-bound AA by free AA could avoid further production of body heat as reported recently (Morales et al., 2019) and prevented additional damage to the intestinal epithelia of HS pigs. However, the question regarding how much more body heat is produced by increasing the protein content in the diet of HS pigs remains to be answered. Furthermore, it is not quite conclusive whether pigs fed the HSaa diet recovered the intestinal epithelia because of a lower intestinal heat production or a direct effect of any of the dietary AA supplied in free form. The increased villi height in the jejunum of HSaa pigs implies an increment in the number of absorptive cells, thus we asked if this response could affect the abundance of AA transporters. Lys and neutral AA transporters are of particular interest because Lys is the first limiting AA in most diets for pigs and because neutral AA, especially Leu, interacts with Lys for absorption. Absorption of Lys in the small intestine involves mainly the activity of two cationic AA transporters, b0,+ and y+L (Broer, 2008), which are expressed in the apical and basolateral membranes of enterocytes, respectively (Torras-Llort et al., 2001). Both transporters function as antiporter exchanging Leu for Lys so that the absorption of Lys is coupled with the availability of Leu (Pineda et al., 2004). System B0 is the major transporter of neutral AA in the small intestine (Broer, 2008); it transports all neutral AA with a high preference for Leu, Ile, Val, and Met (Reimer et al., 2000). In the present experiment, the abundance of mRNA coding for the synthesis of y+L in the jejunum and that of B0 in the ileum of pigs fed the HSaa diet was about 25% and 32%, respectively, of that observed in pigs fed the HShp diet. Similar reductions in the abundance of y+L mRNA (Morales et al., 2015) and B0 mRNA (Cervantes et al., 2017) were observed in the ileum of thermal neutral pigs fed diets supplemented with free AA. Most of Lys in pigs fed the HSaa diet is expected to be absorbed in duodenum because it was contained in free form (Morales et al., 2015). Consequently, a lower amount of dietary Lys (mostly protein-bound) would be left for absorption in jejunum and ileum, which, combined with the increased villi height, may explain the lower intestinal gene expression of AA transporters in the HSaa pigs. The SC of free AA during the absorptive phase reflects the absorption of dietary AA which peaks at about 1.5 to 2.5 h postprandial (Yen et al., 2004; Morales et al., 2016). Protein-bound AA are absorbed mainly in jejunum (Silk et al., 1985) after dietary proteins are digested and the AA are released. However, dietary free AA may be absorbed along the whole intestine including duodenum because these are already free as these enter into the small intestine and because AA transporters are expressed in duodenum, jejunum, and ileum of pigs (Morales et al., 2015) and mice (Dave et al., 2004). Thus, SC of free AA may vary depending on the form dietary AA are supplied. In the present experiment, the HSaa diet contained 56%, 43%, and 42% of free Lys, Met, and Thr, respectively. Therefore, the higher SC of free Lys, Met, and Thr at 2030 hours (1.5 h postprandial) indicates that these AA were rapidly absorbed, which agrees with previous reports (Yen et al., 2004; Morales et al., 2015). Furthermore, the higher SC of Lys in pigs fed the HSaa diet supports the hypothesis that free dietary Lys was mostly absorbed in the proximal small intestine. On the other hand, because all dietary Arg and most of dietary His, Ile, Leu, Phe, Trp, and Val were added as protein-bound AA (only 4 to 13% were added in free form) explains their lack of SC difference at 2030 hours between the HShp and the HSaa pigs. In contrast, the higher SC of most AA at 2130 hours (2.5 h postprandial) in HShp pigs is explained by the higher total content of these AA in the HShp diet. The tendency of Lys SC to be higher at 2130 hours in HSaa pigs may reflect that 1) absorption of Lys from the HShp diet peaks at later than 2.5 h postprandial or 2) the absorbed Lys from the HSaa diet is also utilized more rapidly for body protein accretion. It is worth noting the lack of difference in the SC of Thr between HShp and HSaa pigs, which may be explained by the fact that Thr is the most abundant AA in mucin, whose secretion increases when pigs are exposed to HS. Hence, Thr could become limiting in the HSaa diet suggesting a higher need of Thr for HS pigs fed this type of diet. The SC of AA at 1700 hours (10 h postprandial; postabsorptive values) represents mostly the AA leftover after their removal from serum by the cells (Rérat et al., 1988; Yen et al., 2004). At this sampling time, except for Lys and Met, the higher SC of AA in pigs fed the HShp diet might be attributed to their higher content in comparison with the AA content in the HSaa diet. Both diets contained similar levels of SID Lys and Met but the content of the other indispensable AA was higher in the HShp diet. In addition, it has been reported that high protein content diets reduce the gastric emptying (Cuber and Laplace, 1979) leading to reduced protein digestion and subsequent AA absorption. On the other hand, the reduced Thr SC in pigs fed the HSaa diet may suggest that this AA is limiting in this diet. Interestingly, the SC of free AA in pigs fed the HSaa diet at 2030 hours (1.5 h postprandial) was substantially higher than the preprandial SC regardless of the form in which dietary AA were supplied. In contrast, except for Lys, the SC of the indispensable AA in pigs fed the HShp diet at 1.5 h postprandial did not differ with the preprandial SC. One possible explanation for this difference could be the lower SC of most AA in pigs fed the HSaa diet during the preprandial time, in comparison with the HShp diet. However, it is also possible that this response is due to the improved AA profile in the HSaa diet with no excess or deficiency of AA (ideal protein); the efficiency of utilization of AA appeared to improve as evidenced by the lower urea SC in pigs fed the HSaa diet. Hence, this response may indicate that the combination of dietary free AA with protein-bound AA improves their absorption, availability, and utilization in HS pigs. Growth performance of pigs under thermal neutral condition was sometimes compromised, compared with pigs fed high CP diets by feeding low protein-AA fortified diets which were limiting in the next limiting AA (Otto et al., 2003; Guay and Trottier, 2006). In other studies, however, no difference in performance was observed by lowering the dietary CP level (Yi et al., 2010; Powell et al., 2011). In a previous study, we observed no differences in weight gain and G:F among pigs fed a 14% CP wheat–SBM diet supplemented with free AA and pigs fed a 22% CP diet (Morales et al., 2015). However, knowledge about this type of response in HS pigs was limited. The lack of performance difference observed in the current experiment indicates that the availability of AA did not differ between pigs fed the HShp or the HSaa diet. On the other hand, HS pigs reduce their feed intake by 20% to 40% (Colin et al., 2001; Cervantes et al., 2017), which implies that the consumption of AA decreases in a similar manner. The lowered AA intake resulting from the reduced voluntary feed intake in HS pigs could be corrected by increasing the dietary AA content. Free AA, therefore, could be used to increase the dietary AA content that might eventually improve the performance of HS pigs without the risk of elevating their body heat production. In conclusion, feeding HS pigs with low protein diets supplemented with free AA reduces the damage of the intestinal epithelia and improves its absorption capacity, in comparison with pigs fed diets containing solely protein-bound AA. This information may be useful to formulate diets aiming to compensate the reduced AA consumption associated with the decreased voluntary feed intake of pigs under HS. 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TI - Dietary protein-bound or free amino acids differently affect intestinal morphology, gene expression of amino acid transporters, and serum amino acids of pigs exposed to heat stress
JF - Journal of Animal Science
DO - 10.1093/jas/skaa056
DA - 2020-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/dietary-protein-bound-or-free-amino-acids-differently-affect-tlkg05GcCL
VL - 98
IS - 3
DP - DeepDyve
ER -