DOI: 10.1111/jpn.12327
ORIGINAL ARTICLE
Effect of dietary excess of branched-chain amino acids on performance and serum concentrations of amino acids in growing pigs A. Morales1, N. Arce1, M. Cota1, L. Buenabad1, E. Avelar1, J. K. Htoo2 and M. Cervantes1 noma de Baja California Mexicali, M 1 ICA, Universidad Auto exico, and 2 Nutrition Research, Evonik Industries AG, Hanau, Germany
Summary Depressed performance and availability of some amino acids (AA) in pigs fed excess Leu diets appear to be related to lower feed intake. Surplus Ile and Val may help to overcome this effect. An experiment was conducted with 24 pigs (31.8 1.2 kg initial BW) to evaluate the effect of dietary excess of either Leu alone or with surplus Ile and Val on performance and serum concentration (SC) of essential AA. Treatments were as follows: T1, basal diet; T2, basal plus 0.43% L-Leu (excess Leu); T3, basal added with 0.43% L-Leu, plus 0.20% L-Ile and 0.25% LVal (excess LIV). The basal diet was formulated to contain 0.90% standardized ileal digestible Lys and added with crystalline L-Lys, L-Thr, DL-Met, L-Trp, L-Leu, L-Ile, L-His and L-Val to create essential AA:Lys ratios close to an ideal protein for growing pigs. All pigs were fed the same amount of feed twice a day (average, 3.429 the requirement of NEm). Blood samples were collected at 2.5 (absorptive) and 11.0 h (post-absorptive) post-prandial to analyse SC of AA. Excess of either Leu or LIV did not affect growth rate nor feed conversion. Excess Leu increased Leu SC and decreased Ile and Val SC (p < 0.05) at both absorptive and post-absorptive phases, but excess LIV restored the SC of Ile and Val. The SC of other essential AA was not affected by excess of either Leu or LIV. The SC of all AA during absorptive, on average, was about two times higher than that of post-absorptive phase. These results suggest that the reduced availability (SC) of Ile and Val in pigs consuming excess Leu diets is attributed to a reduced absorption and increased cellular degradation rates of them. Keywords pigs, excess Leu, branched-chain amino acids, amino acid serum concentration, performance noma de Baja California, Av. Alvaro n s/n, CP 21100, Mexicali, BC, Mexico. Correspondence Miguel Cervantes, ICA-Universidad Auto Obrego Tel: +52-686-523 0088; Fax: +52-686-523 0217; E-mail: [email protected] Received: 29 July 2014; accepted: 3 March 2015
Introduction Excess of amino acids (AA) such as Leu in practical high protein diets is common and may negatively affect the availability of the other branched-chain AA (BCAA; Ile and Val) and Lys and consequently depress pig performance. Reduced daily weight gain in weanling (Gatnau et al., 1995; Wiltafsky et al., 2010) and growing (Cervantes-Ramırez et al., 2013) pigs fed excess Leu diets has been associated with a marked increase in serum concentration (SC) of Leu and a decrease in the SC of Ile and Val. Excess dietary Leu increases the activities of branched-chain keto acid dehydrogenase enzymes in liver (Langer et al., 2000; Wiltafsky et al., 2010). Because Ile, Leu and Val share this enzyme complex in their degradative pathways (Langer et al., 2000), excess Leu results in increased
catabolism of all BCAA (Block, 1989) provoking a reduction in the SC of Ile and Val. It has been shown that the reduced SC of AA in pigs fed excess Leu diets was associated with a feed intake (FI) decrease (Gatnau et al., 1995; Wiltafsky et al., 2010), indicating that lower FI was also responsible for the reduced SC of BCAA. Except for Langer et al. (2000) who used semi purified diets, there is no data available showing the effect of excess Leu on the SC of AA and performance of pigs that are fed practical diets and having similar FI level. Interactions between BCAA that share common transport systems for absorption have been reported (Broer, 2008). The major transporter for BCAA (system B0) is expressed along the entire small intestine (Romeo et al., 2006) and equally prefers all three BCAA (Broer, 2008) suggesting that excess Leu may
Journal of Animal Physiology and Animal Nutrition 100 (2016) 39–45 © 2015 Blackwell Verlag GmbH
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Excess of branched amino acids in pigs
impair the absorption of Ile and Val. Hence, excess Leu seems to affect the availability of Ile and Val not only by increasing their cellular degradation but also by reducing their absorption in the small intestine. Excess Leu may also affect the availability of Lys because its transporter (b0,+ AT) exchanges Lys for Leu during absorption (Broer, 2008; Majumder et al., 2009). The intestinal absorption of Lys by b0,+ AT is coupled with the efflux of Leu (Pineda et al., 2004), and excess Leu reduces expression of b0,+ AT in jejunum (Garcıa-Villalobos et al., 2012). Thus, Lys availability may be affected also by excess Leu in the diet. The SC of free AA in pigs comes from both degraded tissue proteins and AA from digested dietary proteins (Adibi and Mercer, 1973). Dietary proteins and AA are the principal source for increased SC of free AA within the first 2.5 h post-prandial or absorptive phase (Yen et al., 2004) and reflect differences in absorbed AA. Likewise, SC of free AA during the postabsorptive phase (8+ h post-prandial) reflects the removal of circulating AA for protein synthesis by the cells (Langer and Fuller, 2000). Thus, SC of AA during the absorptive and post-absorptive phases can be used as indicators of potential AA interactions at the absorption and degradation levels respectively. We hypothesized that, at similar FI level, excess Leu affects the availability of Lys, Ile and Val, and the performance of growing pigs and that the combined surplus of Ile and Val might be necessary to correct that effect. Therefore, this experiment was conducted to evaluate the effect of excess Leu alone or combined with surplus supply of Ile and Val in diets on the SC of AA during both the absorptive and post-absorptive phases, and the performance of growing pigs under similar FI conditions. Materials and methods Animals and diets
Twenty-four cross-bred pigs (Large White 9 Duroc) with initial body weight (BW) of 31.8 1.2 kg) were used in a 21-day growth trial. Pigs were randomly allotted into three dietary treatments. There were eight pig replicates (two barrows and six gilts) per treatment. Pigs were distributed in three treatments based on BW, sex and litter, according to a Complete Block design, and then assigned randomly to pens. 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 nipple water drinker and iron mesh floor in a temperature-controlled room (22–24 °C). One week before the start of the experiment, all pigs were adapted to pens and diets and trained to 40
consume the same amount of feed, which at the end of this week, it was approximately 3.42 times the estimated NEm requirement for pigs with this BW (NRC, 2012). All pigs received this amount of feed during the first week of the experiment, and it was adjusted every week based on the average BW of the pigs. Average daily weight gain (ADG) and feed conversion ratio (FCR) were calculated every week. The growth performance study lasted 21 day and the average BW of the pigs at the end of the trial was 46.8 1.1 kg. The pigs used in this experiment were cared for in accordance with the guidelines established in the Official Mexican Regulations on Animal Care (NOM-062Z00-1999, 2001). A basal diet was formulated with wheat as the sole major feed ingredient, the most common ingredient used under practical conditions in northern M exico, and supplemented with crystalline AA (Table 1). Dietary treatments were as follows: 1, basal diet; 2, basal diet plus 0.43% L-Leu (excess Leu); T3, basal diet added with 0.43% L-Leu, plus 0.20% L-Ile and 0.25% L-Val (excess LIV). The basal diet was formulated to contain 0.90% standardized ileal digestible (SID) Lys using the analysed AA content (Table 2) and published SID coefficient for wheat (Stein et al., 2001). Crystalline L-Lys, L-Thr, DL-Met, L-Trp, L-Leu, L-Ile, L-His and L-Val were added to the basal diet to create essential AA:Lys ratios close to the ideal protein for growing pigs (NRC, 1998). The analysed Ile:Lys, Leu:
Table 1 Composition of the experimental diets (as fed) Dietary treatment
Ingredient, %
Basal
Excess Leu
Excess Leu, Ile, Val
Wheat Corn starch L-Lys • HCl L-Thr DL-Met L-Trp L-Leu L-Ile L-His L-Val Calcium carbonate Orthophosphate Iodized salt Vitamin and mineral premix*
95.00 0.88 0.79 0.27 0.13 0.08 0.10 0.13 0.03 0.16 1. 38 0.40 0.35 0.40
95.00 0.45 0.79 0.27 0.13 0.08 0.53 0.13 0.03 0.16 1. 38 0.40 0.35 0.40
95.00 – 0.79 0.27 0.13 0.08 0.53 0.33 0.03 0.41 1.38 0.40 0.35 0.40
*Supplied per kg of diet: vitamin A, 4800 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.
Journal of Animal Physiology and Animal Nutrition 100 (2016) 39–45 © 2015 Blackwell Verlag GmbH
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Excess of branched amino acids in pigs
Table 2 Analysed composition of the experimental diets (%, as fed) Dietary treatment Amino acid Indispensable Arg His Ile Leu Lys Met Phe Thr Trp Val Dispensable Ala Asp Cys Glu Gly Pro Ser
Basal
Excess Leu
Excess Leu, Ile, Val
0.57 0.29 0.51 0.87 0.88 0.30 0.53 0.56 0.22 0.64
0.57 0.30 0.52 1.23 0.90 0.29 0.54 0.58 0.22 0.66
0.57 0.30 0.70 1.25 0.94 0.30 0.54 0.57 0.21 0.89
0.41 0.62 0.25 3.02 0.43 1.09 0.53
0.42 0.62 0.25 3.09 0.43 1.1 0.53
0.41 0.62 0.24 3.08 0.43 1.11 0.53
Lys and Val:Lys ratios in the basal diet were 58%, 100% and 73% respectively. The addition of L-Leu to diets 2 and 3 increased the dietary Leu content by 42%, as compared to that in the basal diet. Diet 3 contained approximately 45% more Ile and Val, as compared with the basal. All diets were formulated to contain 10.4 MJ NE/kg and supplemented with a mineral–vitamin premix to meet or exceed their requirements for the growing pigs (NRC, 1998). Blood collection
Blood samples from the carotid artery (approximately 10 ml) were collected from eight pigs per treatment, after and before the morning feeding, to analyse the SC of free AA at both the absorptive and post-absorptive states. For the absorptive state, blood samples were collected 2.5 h after the morning meal on d 19 of the study. For the post-absorptive state, blood samples were collected on d 21 at 6:00 hours after pigs were fasted overnight for 11 h. Immediately after collection, all samples were centrifuged at 500 g, 4 °C for 10 min to separate serum from blood cells and stored at 20 °C until analysis. Chemical analyses
The SC of free AA was determined by ion exchange chromatography using a Biochrom 20 amino acid analyser Lithium column and lithium buffers. Amino
acids were oxidized with performic acid, which was neutralized with Na metabisulphite (Llames and Fontaine, 1994). Amino acids were quantified with the internal standard by measuring the absorption of reaction products with ninhydrin at 570 nm. Dietary AA were liberated from protein by hydrolysis with 6 N HCL for 24 h at 110 °C and quantified with the internal standard by measuring the absorption of reaction products with ninhydrin at 570 nm. Tryptophan was determined by HPLC with fluorescence detection (extinction 280 nm, emission 356 nm), after alkaline hydrolysis with barium hydroxide octahydrate for 20 h at 110 °C (Commission Directive, 2000). Statistical analysis
Analyses of variance of the data from each variable were performed using the GLM of SAS (Statistical Analysis System 9.1; SAS Institute, Cary, NC, USA). The following contrasts (C) were constructed to test the effects of excess Leu or excess LIV: C1, Basal vs. excess Leu; C2, excess Leu vs. excess LIV; C3, Basal vs. excess LIV. Probability levels of p ≤ 0.05 and 0.05 < p ≤ 0.10 were defined as significant differences and tendencies respectively. Results Growth performance
The performance results are presented in Table 3. By design, FI was restricted to all pigs to an average of 1.70 kg/day during the whole study. The daily FI per pig during weeks 1, 2 and 3 were 1.60, 1.65 and 1.76 kg respectively. Dietary excess Leu alone did not affect ADG (p = 0.644) and FCR (p = 0.489). Similarly, excess supply of all BCAA (LIV) did not affect the ADG (p = 0.160) and the FCR (p = 0.474). However, pigs fed the excess LIV diet tended to improve ADG (p = 0.069) and FCR (p = 0.060) as compared to their counterparts fed the excess Leu diet. Concentration of free amino acids in serum Absorptive phase
Serum concentrations of BCAA were differently affected by the dietary excesses of Leu alone or LIV in the basal diet (Table 4). Excess Leu decreased SC of Ile (p = 0.015) and Val (p = 0.001), but increased that of Leu (p = 0.017). Excess LIV, as compared with the basal diet, did not affect the SC of Ile (p = 0.897) and Val (p = 0.599) but increased SC of Leu (p = 0.034) and Lys (p = 0.020). However, when compared to excess Leu, excess LIV increased the SC of Ile
Journal of Animal Physiology and Animal Nutrition 100 (2016) 39–45 © 2015 Blackwell Verlag GmbH
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Excess of branched amino acids in pigs
Dietary treatment*
Contrasts p value†
Item
Basal
Excess Leu
Excess LIV
SEM
1
2
3
Initial weight, kg Final weight, kg Average daily gain, g/d Feed intake, kg/d Feed conversion ratio
31.7 46.5 707 1.68 2.23
31.9 46.6 698 1.70 2.30
31.8 47.3 737 1.69 2.16
1.13 14.3 0.01 0.07
0.929 0.644 0.195 0.489
0.630 0.069 0.485 0.060
0.469 0.160 0.530 0.474
Table 3 Effect of dietary excess Leu without or with excess Ile and Val on the average daily gain, daily feed intake and feed conversion rate of growing pigs fed a wheat-based diet
*Basal: wheat + L-Lys, L-Thr, DL-Met, L-Trp, L-Leu, L-Ile, L-His, L-Val; Excess Leu: Basal + excess Leu; Excess LIV: Basal Excess LIV. †Contrasts: 1, Basal vs. Excess Leu; 2, Excess Leu vs. Excess LIV; 3, Basal vs. Excess LIV.
Table 4 Effect of dietary excess Leu without or with excess Ile and Val on the serum concentration of AA (mg/100 ml) during the absorptive phase of pigs fed a wheat-based diet Dietary treatment* Excess amino acid Amino acid
Basal
Indispensable Arg 3.82 His 0.95 Ile 3.15 Leu 3.02 Lys 5.09 Met 1.36 Phe 1.25 Thr 4.44 Val 8.14 Dispensable Ala 8.12 Asn 0.62 Asp 0.22 Cys 0.65 Gln 8.99 Glu 2.23 Gly 7.54 Pro 6.95 Ser 2.23 Tyr 1.18
Excess Leu
Excess LIV
Contrasts p value†
SEM
1
2
3
3.92 0.95 1.38 4.62 5.62 1.15 1.36 4.60 3.71
4.07 1.02 3.47 4.90 7.87 1.34 1.50 5.64 8.65
0.46 0.23 0.42 0.45 0.69 0.19 0.16 0.62 0.66
0.883 0.991 0.015 0.017 0.607 0.466 0.647 0.862 0.001
0.827 0.846 0.006 0.679 0.047 0.505 0.560 0.267 0.10). The dispensable AA, except for Ser that tended to decrease (p = 0.090), were not affected by 42
excess Leu (p > 0.10). Excess LIV, as compared to excess Leu, increased Ala (p = 0.005) and Ser (p = 0.020), tended to increase Asn (p = 0.092) and Glu (p = 0.096), but did not affect the remaining dispensable AA (p > 0.10). Compared to the basal diet, excess LIV increased SC of Ala (p = 0.022) and tended to increase Asn (p = 0.089) and Cys (p = 0.065). Post-absorptive phase
The SC of the BCAA was also differently affected by the dietary excesses of Leu or LIV in the basal diet (Table 5). Pigs fed the excess Leu diet had lower SC of Ile and Val (p < 0.001) but higher Leu (p = 0.009) than pigs fed the basal diet. The SC of Ile and Val in pigs fed excess LIV was higher (p < 0.001) than in pigs fed the excess Leu diet, but serum Leu was not different (p = 0.662). Feeding the excess LIV diet to pigs increased the SC of Leu (p = 0.004) and lowered the SC of Val (p = 0.050), but did not affect the SC of Ile (p = 0.166), in comparison with that in pigs fed the basal diet. The SC of dispensable AA was not affected by the dietary excess Leu or the excess LIV (p > 0.10). Discussion Reductions in both SC of some AA and growth rate of pigs fed excess Leu diets are often associated with lower FI (Gatnau et al., 1995; Wiltafsky et al., 2010), which result in decreased ingestion of nutrients compared with pigs fed normal Leu diets. All pigs in the present study were fed the same amount of feed to eliminate any potential effect that differences in FI might have on nutrient consumption, other than Leu, Ile and Val. Pigs fed the excess Leu diet consumed approximately 42% more Leu than those fed the basal, but there were no differences in the consumption of other nutrients. Likewise pigs fed the excess LIV diet consumed 42%, 20% and 25% more Leu, Ile
Journal of Animal Physiology and Animal Nutrition 100 (2016) 39–45 © 2015 Blackwell Verlag GmbH
A. Morales et al.
Excess of branched amino acids in pigs
Table 5 Effect of dietary excess Leu with or without excess Ile and Val on the serum concentration of AA (mg/100 ml) during the post-absorptive phase of pigs fed a wheat-based diet formulated on the basis of the ideal protein concept Dietary treatment* Excess amino acid Amino acid
Basal
Indispensable Arg 2.07 His 0.33 Ile 1.92 Leu 1.80 Lys 1.89 Met 0.47 Phe 0.84 Thr 2.15 Val 5.58 Dispensable Ala 6.50 Asn 0.49 Asp 0.22 Cys 0.59 Gln 8.90 Glu 2.07 Gly 7.06 Pro 3.12 Ser 1.41 Tyr 0.65
Contrasts p value†
Excess Leu
Excess LIV
SEM
1
2
3
2.45 0.47 0.91 2.63 1.38 0.42 0.97 2.72 2.35
2.07 0.53 1.73 2.74 1.88 0.49 0.94 2.18 4.91
0.17 0.11 0.09 0.17 0.42 0.06 0.09 0.28 0.21
0.146 0.389
ORIGINAL ARTICLE
Effect of dietary excess of branched-chain amino acids on performance and serum concentrations of amino acids in growing pigs A. Morales1, N. Arce1, M. Cota1, L. Buenabad1, E. Avelar1, J. K. Htoo2 and M. Cervantes1 noma de Baja California Mexicali, M 1 ICA, Universidad Auto exico, and 2 Nutrition Research, Evonik Industries AG, Hanau, Germany
Summary Depressed performance and availability of some amino acids (AA) in pigs fed excess Leu diets appear to be related to lower feed intake. Surplus Ile and Val may help to overcome this effect. An experiment was conducted with 24 pigs (31.8 1.2 kg initial BW) to evaluate the effect of dietary excess of either Leu alone or with surplus Ile and Val on performance and serum concentration (SC) of essential AA. Treatments were as follows: T1, basal diet; T2, basal plus 0.43% L-Leu (excess Leu); T3, basal added with 0.43% L-Leu, plus 0.20% L-Ile and 0.25% LVal (excess LIV). The basal diet was formulated to contain 0.90% standardized ileal digestible Lys and added with crystalline L-Lys, L-Thr, DL-Met, L-Trp, L-Leu, L-Ile, L-His and L-Val to create essential AA:Lys ratios close to an ideal protein for growing pigs. All pigs were fed the same amount of feed twice a day (average, 3.429 the requirement of NEm). Blood samples were collected at 2.5 (absorptive) and 11.0 h (post-absorptive) post-prandial to analyse SC of AA. Excess of either Leu or LIV did not affect growth rate nor feed conversion. Excess Leu increased Leu SC and decreased Ile and Val SC (p < 0.05) at both absorptive and post-absorptive phases, but excess LIV restored the SC of Ile and Val. The SC of other essential AA was not affected by excess of either Leu or LIV. The SC of all AA during absorptive, on average, was about two times higher than that of post-absorptive phase. These results suggest that the reduced availability (SC) of Ile and Val in pigs consuming excess Leu diets is attributed to a reduced absorption and increased cellular degradation rates of them. Keywords pigs, excess Leu, branched-chain amino acids, amino acid serum concentration, performance noma de Baja California, Av. Alvaro n s/n, CP 21100, Mexicali, BC, Mexico. Correspondence Miguel Cervantes, ICA-Universidad Auto Obrego Tel: +52-686-523 0088; Fax: +52-686-523 0217; E-mail: [email protected] Received: 29 July 2014; accepted: 3 March 2015
Introduction Excess of amino acids (AA) such as Leu in practical high protein diets is common and may negatively affect the availability of the other branched-chain AA (BCAA; Ile and Val) and Lys and consequently depress pig performance. Reduced daily weight gain in weanling (Gatnau et al., 1995; Wiltafsky et al., 2010) and growing (Cervantes-Ramırez et al., 2013) pigs fed excess Leu diets has been associated with a marked increase in serum concentration (SC) of Leu and a decrease in the SC of Ile and Val. Excess dietary Leu increases the activities of branched-chain keto acid dehydrogenase enzymes in liver (Langer et al., 2000; Wiltafsky et al., 2010). Because Ile, Leu and Val share this enzyme complex in their degradative pathways (Langer et al., 2000), excess Leu results in increased
catabolism of all BCAA (Block, 1989) provoking a reduction in the SC of Ile and Val. It has been shown that the reduced SC of AA in pigs fed excess Leu diets was associated with a feed intake (FI) decrease (Gatnau et al., 1995; Wiltafsky et al., 2010), indicating that lower FI was also responsible for the reduced SC of BCAA. Except for Langer et al. (2000) who used semi purified diets, there is no data available showing the effect of excess Leu on the SC of AA and performance of pigs that are fed practical diets and having similar FI level. Interactions between BCAA that share common transport systems for absorption have been reported (Broer, 2008). The major transporter for BCAA (system B0) is expressed along the entire small intestine (Romeo et al., 2006) and equally prefers all three BCAA (Broer, 2008) suggesting that excess Leu may
Journal of Animal Physiology and Animal Nutrition 100 (2016) 39–45 © 2015 Blackwell Verlag GmbH
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Excess of branched amino acids in pigs
impair the absorption of Ile and Val. Hence, excess Leu seems to affect the availability of Ile and Val not only by increasing their cellular degradation but also by reducing their absorption in the small intestine. Excess Leu may also affect the availability of Lys because its transporter (b0,+ AT) exchanges Lys for Leu during absorption (Broer, 2008; Majumder et al., 2009). The intestinal absorption of Lys by b0,+ AT is coupled with the efflux of Leu (Pineda et al., 2004), and excess Leu reduces expression of b0,+ AT in jejunum (Garcıa-Villalobos et al., 2012). Thus, Lys availability may be affected also by excess Leu in the diet. The SC of free AA in pigs comes from both degraded tissue proteins and AA from digested dietary proteins (Adibi and Mercer, 1973). Dietary proteins and AA are the principal source for increased SC of free AA within the first 2.5 h post-prandial or absorptive phase (Yen et al., 2004) and reflect differences in absorbed AA. Likewise, SC of free AA during the postabsorptive phase (8+ h post-prandial) reflects the removal of circulating AA for protein synthesis by the cells (Langer and Fuller, 2000). Thus, SC of AA during the absorptive and post-absorptive phases can be used as indicators of potential AA interactions at the absorption and degradation levels respectively. We hypothesized that, at similar FI level, excess Leu affects the availability of Lys, Ile and Val, and the performance of growing pigs and that the combined surplus of Ile and Val might be necessary to correct that effect. Therefore, this experiment was conducted to evaluate the effect of excess Leu alone or combined with surplus supply of Ile and Val in diets on the SC of AA during both the absorptive and post-absorptive phases, and the performance of growing pigs under similar FI conditions. Materials and methods Animals and diets
Twenty-four cross-bred pigs (Large White 9 Duroc) with initial body weight (BW) of 31.8 1.2 kg) were used in a 21-day growth trial. Pigs were randomly allotted into three dietary treatments. There were eight pig replicates (two barrows and six gilts) per treatment. Pigs were distributed in three treatments based on BW, sex and litter, according to a Complete Block design, and then assigned randomly to pens. 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 nipple water drinker and iron mesh floor in a temperature-controlled room (22–24 °C). One week before the start of the experiment, all pigs were adapted to pens and diets and trained to 40
consume the same amount of feed, which at the end of this week, it was approximately 3.42 times the estimated NEm requirement for pigs with this BW (NRC, 2012). All pigs received this amount of feed during the first week of the experiment, and it was adjusted every week based on the average BW of the pigs. Average daily weight gain (ADG) and feed conversion ratio (FCR) were calculated every week. The growth performance study lasted 21 day and the average BW of the pigs at the end of the trial was 46.8 1.1 kg. The pigs used in this experiment were cared for in accordance with the guidelines established in the Official Mexican Regulations on Animal Care (NOM-062Z00-1999, 2001). A basal diet was formulated with wheat as the sole major feed ingredient, the most common ingredient used under practical conditions in northern M exico, and supplemented with crystalline AA (Table 1). Dietary treatments were as follows: 1, basal diet; 2, basal diet plus 0.43% L-Leu (excess Leu); T3, basal diet added with 0.43% L-Leu, plus 0.20% L-Ile and 0.25% L-Val (excess LIV). The basal diet was formulated to contain 0.90% standardized ileal digestible (SID) Lys using the analysed AA content (Table 2) and published SID coefficient for wheat (Stein et al., 2001). Crystalline L-Lys, L-Thr, DL-Met, L-Trp, L-Leu, L-Ile, L-His and L-Val were added to the basal diet to create essential AA:Lys ratios close to the ideal protein for growing pigs (NRC, 1998). The analysed Ile:Lys, Leu:
Table 1 Composition of the experimental diets (as fed) Dietary treatment
Ingredient, %
Basal
Excess Leu
Excess Leu, Ile, Val
Wheat Corn starch L-Lys • HCl L-Thr DL-Met L-Trp L-Leu L-Ile L-His L-Val Calcium carbonate Orthophosphate Iodized salt Vitamin and mineral premix*
95.00 0.88 0.79 0.27 0.13 0.08 0.10 0.13 0.03 0.16 1. 38 0.40 0.35 0.40
95.00 0.45 0.79 0.27 0.13 0.08 0.53 0.13 0.03 0.16 1. 38 0.40 0.35 0.40
95.00 – 0.79 0.27 0.13 0.08 0.53 0.33 0.03 0.41 1.38 0.40 0.35 0.40
*Supplied per kg of diet: vitamin A, 4800 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.
Journal of Animal Physiology and Animal Nutrition 100 (2016) 39–45 © 2015 Blackwell Verlag GmbH
A. Morales et al.
Excess of branched amino acids in pigs
Table 2 Analysed composition of the experimental diets (%, as fed) Dietary treatment Amino acid Indispensable Arg His Ile Leu Lys Met Phe Thr Trp Val Dispensable Ala Asp Cys Glu Gly Pro Ser
Basal
Excess Leu
Excess Leu, Ile, Val
0.57 0.29 0.51 0.87 0.88 0.30 0.53 0.56 0.22 0.64
0.57 0.30 0.52 1.23 0.90 0.29 0.54 0.58 0.22 0.66
0.57 0.30 0.70 1.25 0.94 0.30 0.54 0.57 0.21 0.89
0.41 0.62 0.25 3.02 0.43 1.09 0.53
0.42 0.62 0.25 3.09 0.43 1.1 0.53
0.41 0.62 0.24 3.08 0.43 1.11 0.53
Lys and Val:Lys ratios in the basal diet were 58%, 100% and 73% respectively. The addition of L-Leu to diets 2 and 3 increased the dietary Leu content by 42%, as compared to that in the basal diet. Diet 3 contained approximately 45% more Ile and Val, as compared with the basal. All diets were formulated to contain 10.4 MJ NE/kg and supplemented with a mineral–vitamin premix to meet or exceed their requirements for the growing pigs (NRC, 1998). Blood collection
Blood samples from the carotid artery (approximately 10 ml) were collected from eight pigs per treatment, after and before the morning feeding, to analyse the SC of free AA at both the absorptive and post-absorptive states. For the absorptive state, blood samples were collected 2.5 h after the morning meal on d 19 of the study. For the post-absorptive state, blood samples were collected on d 21 at 6:00 hours after pigs were fasted overnight for 11 h. Immediately after collection, all samples were centrifuged at 500 g, 4 °C for 10 min to separate serum from blood cells and stored at 20 °C until analysis. Chemical analyses
The SC of free AA was determined by ion exchange chromatography using a Biochrom 20 amino acid analyser Lithium column and lithium buffers. Amino
acids were oxidized with performic acid, which was neutralized with Na metabisulphite (Llames and Fontaine, 1994). Amino acids were quantified with the internal standard by measuring the absorption of reaction products with ninhydrin at 570 nm. Dietary AA were liberated from protein by hydrolysis with 6 N HCL for 24 h at 110 °C and quantified with the internal standard by measuring the absorption of reaction products with ninhydrin at 570 nm. Tryptophan was determined by HPLC with fluorescence detection (extinction 280 nm, emission 356 nm), after alkaline hydrolysis with barium hydroxide octahydrate for 20 h at 110 °C (Commission Directive, 2000). Statistical analysis
Analyses of variance of the data from each variable were performed using the GLM of SAS (Statistical Analysis System 9.1; SAS Institute, Cary, NC, USA). The following contrasts (C) were constructed to test the effects of excess Leu or excess LIV: C1, Basal vs. excess Leu; C2, excess Leu vs. excess LIV; C3, Basal vs. excess LIV. Probability levels of p ≤ 0.05 and 0.05 < p ≤ 0.10 were defined as significant differences and tendencies respectively. Results Growth performance
The performance results are presented in Table 3. By design, FI was restricted to all pigs to an average of 1.70 kg/day during the whole study. The daily FI per pig during weeks 1, 2 and 3 were 1.60, 1.65 and 1.76 kg respectively. Dietary excess Leu alone did not affect ADG (p = 0.644) and FCR (p = 0.489). Similarly, excess supply of all BCAA (LIV) did not affect the ADG (p = 0.160) and the FCR (p = 0.474). However, pigs fed the excess LIV diet tended to improve ADG (p = 0.069) and FCR (p = 0.060) as compared to their counterparts fed the excess Leu diet. Concentration of free amino acids in serum Absorptive phase
Serum concentrations of BCAA were differently affected by the dietary excesses of Leu alone or LIV in the basal diet (Table 4). Excess Leu decreased SC of Ile (p = 0.015) and Val (p = 0.001), but increased that of Leu (p = 0.017). Excess LIV, as compared with the basal diet, did not affect the SC of Ile (p = 0.897) and Val (p = 0.599) but increased SC of Leu (p = 0.034) and Lys (p = 0.020). However, when compared to excess Leu, excess LIV increased the SC of Ile
Journal of Animal Physiology and Animal Nutrition 100 (2016) 39–45 © 2015 Blackwell Verlag GmbH
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A. Morales et al.
Excess of branched amino acids in pigs
Dietary treatment*
Contrasts p value†
Item
Basal
Excess Leu
Excess LIV
SEM
1
2
3
Initial weight, kg Final weight, kg Average daily gain, g/d Feed intake, kg/d Feed conversion ratio
31.7 46.5 707 1.68 2.23
31.9 46.6 698 1.70 2.30
31.8 47.3 737 1.69 2.16
1.13 14.3 0.01 0.07
0.929 0.644 0.195 0.489
0.630 0.069 0.485 0.060
0.469 0.160 0.530 0.474
Table 3 Effect of dietary excess Leu without or with excess Ile and Val on the average daily gain, daily feed intake and feed conversion rate of growing pigs fed a wheat-based diet
*Basal: wheat + L-Lys, L-Thr, DL-Met, L-Trp, L-Leu, L-Ile, L-His, L-Val; Excess Leu: Basal + excess Leu; Excess LIV: Basal Excess LIV. †Contrasts: 1, Basal vs. Excess Leu; 2, Excess Leu vs. Excess LIV; 3, Basal vs. Excess LIV.
Table 4 Effect of dietary excess Leu without or with excess Ile and Val on the serum concentration of AA (mg/100 ml) during the absorptive phase of pigs fed a wheat-based diet Dietary treatment* Excess amino acid Amino acid
Basal
Indispensable Arg 3.82 His 0.95 Ile 3.15 Leu 3.02 Lys 5.09 Met 1.36 Phe 1.25 Thr 4.44 Val 8.14 Dispensable Ala 8.12 Asn 0.62 Asp 0.22 Cys 0.65 Gln 8.99 Glu 2.23 Gly 7.54 Pro 6.95 Ser 2.23 Tyr 1.18
Excess Leu
Excess LIV
Contrasts p value†
SEM
1
2
3
3.92 0.95 1.38 4.62 5.62 1.15 1.36 4.60 3.71
4.07 1.02 3.47 4.90 7.87 1.34 1.50 5.64 8.65
0.46 0.23 0.42 0.45 0.69 0.19 0.16 0.62 0.66
0.883 0.991 0.015 0.017 0.607 0.466 0.647 0.862 0.001
0.827 0.846 0.006 0.679 0.047 0.505 0.560 0.267 0.10). The dispensable AA, except for Ser that tended to decrease (p = 0.090), were not affected by 42
excess Leu (p > 0.10). Excess LIV, as compared to excess Leu, increased Ala (p = 0.005) and Ser (p = 0.020), tended to increase Asn (p = 0.092) and Glu (p = 0.096), but did not affect the remaining dispensable AA (p > 0.10). Compared to the basal diet, excess LIV increased SC of Ala (p = 0.022) and tended to increase Asn (p = 0.089) and Cys (p = 0.065). Post-absorptive phase
The SC of the BCAA was also differently affected by the dietary excesses of Leu or LIV in the basal diet (Table 5). Pigs fed the excess Leu diet had lower SC of Ile and Val (p < 0.001) but higher Leu (p = 0.009) than pigs fed the basal diet. The SC of Ile and Val in pigs fed excess LIV was higher (p < 0.001) than in pigs fed the excess Leu diet, but serum Leu was not different (p = 0.662). Feeding the excess LIV diet to pigs increased the SC of Leu (p = 0.004) and lowered the SC of Val (p = 0.050), but did not affect the SC of Ile (p = 0.166), in comparison with that in pigs fed the basal diet. The SC of dispensable AA was not affected by the dietary excess Leu or the excess LIV (p > 0.10). Discussion Reductions in both SC of some AA and growth rate of pigs fed excess Leu diets are often associated with lower FI (Gatnau et al., 1995; Wiltafsky et al., 2010), which result in decreased ingestion of nutrients compared with pigs fed normal Leu diets. All pigs in the present study were fed the same amount of feed to eliminate any potential effect that differences in FI might have on nutrient consumption, other than Leu, Ile and Val. Pigs fed the excess Leu diet consumed approximately 42% more Leu than those fed the basal, but there were no differences in the consumption of other nutrients. Likewise pigs fed the excess LIV diet consumed 42%, 20% and 25% more Leu, Ile
Journal of Animal Physiology and Animal Nutrition 100 (2016) 39–45 © 2015 Blackwell Verlag GmbH
A. Morales et al.
Excess of branched amino acids in pigs
Table 5 Effect of dietary excess Leu with or without excess Ile and Val on the serum concentration of AA (mg/100 ml) during the post-absorptive phase of pigs fed a wheat-based diet formulated on the basis of the ideal protein concept Dietary treatment* Excess amino acid Amino acid
Basal
Indispensable Arg 2.07 His 0.33 Ile 1.92 Leu 1.80 Lys 1.89 Met 0.47 Phe 0.84 Thr 2.15 Val 5.58 Dispensable Ala 6.50 Asn 0.49 Asp 0.22 Cys 0.59 Gln 8.90 Glu 2.07 Gly 7.06 Pro 3.12 Ser 1.41 Tyr 0.65
Contrasts p value†
Excess Leu
Excess LIV
SEM
1
2
3
2.45 0.47 0.91 2.63 1.38 0.42 0.97 2.72 2.35
2.07 0.53 1.73 2.74 1.88 0.49 0.94 2.18 4.91
0.17 0.11 0.09 0.17 0.42 0.06 0.09 0.28 0.21
0.146 0.389
