The optimum ratio of standardized ileal digestible isoleucine to lysine for 8–15 kg pigs

Animal Feed Science and Technology 198 (2014) 158–165

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The optimum ratio of standardized ileal digestible isoleucine to lysine for 8–15 kg pigs E.A. Soumeh a , J. van Milgen b , N.M. Sloth c , E. Corrent d , H.D. Poulsen a , J.V. Nørgaard a,∗ a b c d

Department of Animal Science, Aarhus University, Foulum, DK-8830 Tjele, Denmark INRA, UMR1348 PEGASE, 35590 Rennes, France Pig Research Centre, Agro Food Park 15, DK-8200 Aarhus N, Denmark Ajinomoto Eurolysine s.a.s., 75817 Paris Cedex 17, France

a r t i c l e

i n f o

Article history: Received 19 May 2014 Received in revised form 30 July 2014 Accepted 12 September 2014 Keywords: Isoleucine Lysine Pigs Performance

a b s t r a c t Research on AA requirements and their profile is still running and the recommendations are being updated frequently. The dietary crude protein level could be reduced with no marked detrimental effects on animal performance as far as the supply and the balance of indispensable AA meet the animal requirements. A dose-response experiment was conducted to estimate the standardized ileal digestible (SID) isoleucine (Ile) to lysine (Lys) ratio required for the best performance of animals when fed cereal-based diets without blood products. In this study, 96 pigs (initial BW 8 kg) were allotted to 1 of 6 dietary treatments with 16 pigs per treatment. Graded levels of crystalline Ile were added to the basal diet to produce diets providing 0.42, 0.46, 0.50, 0.58, and 0.62 SID Ile:Lys. Average daily feed intake (ADFI), average daily gain (ADG), and feed conversion ratio (FCR) were determined during a period of 14 days. Blood and urine samples were taken at the end of each week. There was a quadratic increase in ADFI (P<0.001) and ADG (P<0.007) by increasing level of Ile:Lys in the diet. The ADG tended to increase linearly (P<0.08) as well. The maximum ADFI and ADG were obtained in pigs fed the 0.50 SID Ile:Lys diet. The FCR showed neither linear nor quadratic response to increasing concentration of Ile:Lys in the diet, but numerically the 0.58 and 0.62 Ile:Lys resulted in the lowest FCR. Increasing the dietary SID Ile:Lys resulted in a linear increase in plasma Ile concentration (P<0.001) and a quadratic decrease in plasma leucine concentration (P<0.03). There was also a linear decrease in plasma glycine (P<0.001) and serine (P<0.004) concentrations when increasing dietary Ile:Lys. Neither plasma urea nor urinary urea were affected by feeding the SID Ile:Lys levels. The minimum SID Ile:Lys levels required to maximize ADFI and ADG were 0.51 and 0.52 SID Ile:Lys using a curvilinear plateau model and 0.53 and 0.53 SID Ile:Lys using a quadratic regression model. The estimated requirement using FCR as a response variable was 0.48 SID Ile:Lys by a broken-line model. In conclusion, the optimum SID Ile:Lys in the present experiment was 0.52 in order to maximize ADFI and ADG and 0.48 in order to minimize FCR for 8–15 kg pigs. © 2014 Elsevier B.V. All rights reserved.

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (J.V. Nørgaard). http://dx.doi.org/10.1016/j.anifeedsci.2014.09.013 0377-8401/© 2014 Elsevier B.V. All rights reserved.

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1. Introduction The interest to reduce the crude protein content in diets for pigs regarding health issues and environmental concerns provides the basis for more research on the ideal protein concept. The ideal protein concept defines the profile of indispensable amino acids (AA) which covers the need for individual AA to support maintenance and production. Isoleucine (Ile) along with the other branched-chain AA (BCAA) is one of the indispensable AA, and previous studies reported that in corn–soybean meal diets for pigs, Ile, histidine (His), valine (Val), and tryptophan are equally limiting after lysine (Lys), methionine and threonine (Brudevold and Southern, 1994; Mavromichalis et al., 1998). There is a limited number of studies reporting the Ile requirements in cereal-based diets without using blood products for growing pigs (Van Milgen et al., 2012), and most of the experiments on the determination of Ile requirements have used blood cells in the diet (Kerr et al., 2004a,b; Wiltafsky et al., 2009). Blood products contain high leucine (Leu), Val, His, and phenylalanine (Phe) but low Ile contents. This imbalance increases Ile requirement due to the BCAA interactions between Ile and Leu. Also high His and Phe contents contribute to the increased Ile requirement (Van Milgen et al., 2012). The interaction among BCAA is due to the same enzymes catalyzing the first two steps of BCAA catabolism, and therefore, the excess of one BCAA will result in the catabolism of another (Harper et al., 1984). On the other hand, the large neutral AA (LNAA), His and Phe, are being transported across the blood–brain barrier by the same transporter as Ile, Leu, Val, tryptophan, tyrosine, methionine, threonine, and glutamine (Smith, 2000). The affinity of the transporter to BCAA and LNAA differs and it is much greater for Leu (7-fold) and Phe (2.5-fold) than Ile. Therefore, the excess of these AA would impair Ile transport through blood–brain barrier (Smith, 2000). Thus, the Ile requirement is higher in diets containing blood products as the protein source (Van Milgen et al., 2012). The current recommendations of Ile requirement for growth in pigs (relative to Lys) are 0.52 (NRC, 2012), 0.53 (Tybirk et al., 2012), 0.52 (Gloaguen et al., 2013) and 0.58 (BSAS, 2003). The objective of the current study was to estimate the standardized ileal digestible (SID) Ile requirement in ratio to Lys in pig weighing 8–15 kg in diets based on wheat, barley, and soy protein concentrate.

2. Materials and methods The experiment complied with the Danish Ministry of Justice, Law no. 253 of March 8 2013 concerning experiments with animals and care of experimental animals, and a license issued by the Danish Animal Experiments Inspectorate.

2.1. Animals and diets A total of 96 cross-bred (Danish Landrace, Yorkshire × Duroc) female pigs were individually housed in 1 × 2.2 m pens with one-third concrete floor and two-third cast iron slatted floor which were placed in 4 identical rooms. The temperature and humidity were kept around 22 ◦ C and 60%, respectively, during the experimental period. Diets based on wheat, barley, and soy protein concentrate and were formulated to be isonitrogenous (163 g CP/kg diet) and isoenergetic (10.5 MJ NE/kg; Table 1). The experimental diets were analyzed prior to the experiment. The diets were formulated to contain 0.42, 0.46, 0.50, 0.54, 0.58, and 0.62 of SID Ile:Lys, with Lys being sub-limiting at 93% of Lys requirements (equal to 12.3 g/kg total Lys or 11.4 g SID Lys/kg) (Tybirk et al., 2012). The SID ratios of tryptophan and Val relative to Lys were 0.22 and 0.70, respectively, and the other essential AA as well as calcium and phosphorus were supplied according to Danish recommendations for pigs weighing 9–15 kg (Tybirk et al., 2012). The analyzed and calculated composition of the experimental diets is presented in Table 2.

2.2. Experimental design The experiment was conducted in 2 replicates, with a duration of 2 weeks each. Pigs were weaned at 28 days of age, and the experiments started 1 week after weaning with an average initial body weight of 8.9 ± 0.6 kg (mean ± SD). To determine the minimum SID Ile:Lys supply to maximize performance, a dose-response experiment was conducted. Pigs were allotted individually to 1 of the 6 experimental diets having ad libitum access to feed and water and were weighed on days 7 and 14 without preceding fasting. Average daily feed intake (ADFI) and average daily gain (ADG) were recorded at the end of each week. At days 8 and 15 of the experiment, after overnight fasting, pigs were supplied with 25 g/kg BW0.75 of feed at 0700 h, and blood and urine samples were taken 3 h later from 8 pigs per treatment. Blood samples were collected by jugular vein puncture into 10 ml heparinized tubes (Greiner BioOne GmbH, Kremsmünster, Austria) and placed on ice. Blood samples were centrifuged at 3000 × g at 4 ◦ C for 10 min, and the plasma was immediately harvested and stored at −80 ◦ C until the laboratory analysis. Urine samples were collected using tampons covered by cotton pads which were mounted to the back of pigs with surgical tape at the time of blood collection. Pigs were monitored daily for health condition and were treated with antibiotics (Oxytetracyclin, Engemycin; MSD Animal Health, Willington, New Zealand) in the case of diarrhea.

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Table 1 Composition of experimental diets (as-fed basis). Ingredients, g/kg

Wheat Barley Soy protein concentrateb Animal fat Calcium carbonate Monocalcium phosphate Salt Vitamin–mineral premixc l-Lysine HCl (78%) dl-Methionine (99%) l-Threonine (98%) l-Tryptophan (40%) l-Valine (99%) l-Leucine (98%) l-Isoleucine (98%) l-Histidine (98%) l-Phenylalanine (98%) l-Tyrosine (98%) l-Glutamine (98%)d Phytasee Microgritsf

SIDa Ile:Lys 0.42

0.46

0.50

0.54

0.58

0.62

705.6 100.0 106.5 20.0 16.6 13.3 4.4 4.0 7.9 2.5 3.5 1.1 3.2 3.8 0.0 1.0 1.7 1.4 2.5 0.2 0.7

705.6 100.0 106.5 20.0 16.6 13.3 4.4 4.0 7.9 2.5 3.5 1.1 3.2 3.8 0.5 1.0 1.7 1.4 2.0 0.2 0.7

705.6 100.0 106.5 20.0 16.6 13.3 4.4 4.0 7.9 2.5 3.5 1.1 3.2 3.8 0.9 1.0 1.7 1.4 1.6 0.2 0.7

705.6 100.0 106.5 20.0 16.6 13.3 4.4 4.0 7.9 2.5 3.5 1.1 3.2 3.8 1.4 1.0 1.7 1.4 1.1 0.2 0.7

705.6 100.0 106.5 20.0 16.6 13.3 4.4 4.0 7.9 2.5 3.5 1.1 3.2 3.8 1.9 1.0 1.7 1.4 0.6 0.2 0.7

705.6 100.0 106.5 20.0 16.6 13.3 4.4 4.0 7.9 2.5 3.5 1.1 3.2 3.8 2.3 1.0 1.7 1.4 0.2 0.2 0.7

a

SID = standardized ileal digestible isoleucine to lysine ratio. HP300 (Hamlet Protein, Horsens, Denmark). c Provided the following per kg of diet: 10,000 IU vitamin A, 2000 IU vitamin D3 , 94 IU vitamin E, 2.4 mg vitamin K3 , 2.4 mg vitamin B1 , 4.8 mg vitamin B2 , 2.4 mg vitamin B6 , 0.02 mg vitamin B12 , 12 mg D-panthotenic acid, 26 mg niacin, 0.2 mg biotin, 200 mg Fe (Fe(II) sulphate), 165 mg Cu (Cu(II) sulphate), 200 mg Zn (Zn(II) oxide), 56 mg Mn (Mn(II) oxide), 0.3 mg KI, 0.3 mg Se (Se-selenite). d Included to compose isonitrogenic diets. e Natuphos 5000 (BASF, Ludwigshafen, Germany). f Jadis Additiva (Haarlem, NL). Corn bran in various colors to identify diets. b

2.3. Chemical analysis Nitrogen content was analyzed by a modified Kjeldahl method (AOAC, 2000), and CP content was estimated as total nitrogen × 6.25. Representative samples (n = 3) of each diet were hydrolyzed for 23 h at 110 ◦ C with (for cystine and methionine) or without (for all other AA) performic acid oxidation, and AA were separated by ion exchange chromatography and quantified by photometric detection after ninhydrin reaction (European Commission, 1998). Plasma free AA and urea were analyzed using an AA analyzer fitted to a lithium high performance system for physiological AA (Biochrome 30+ Amino Acid Analyzer; Biochrome, Cambridge, England). The AA analyzer was calibrated using a standard for acidic, neutral, and basic AA (Sigma Aldrich, St. Louis, MO). Urine urea nitrogen and creatinine were determined according to standard procedures (Siemens Diagnostics Clinical Methods) by using an autoanalyzer, ADVIA 1650 Chemistry System (Siemens Medical Solutions, Tarrytown, NY 10591, USA). 2.4. Calculations and statistical analysis Feed conversion ratio (FCR) was calculated by ADFI divided by ADG. The urea content of the urine was divided by creatinine content to provide the urea to creatinine ratio (Kaneko et al., 1998). The data were analyzed by the MIXED procedure of SAS (Version 9.3, SAS Inst. Inc., Cary, NC). The experimental unit was the individual pig. The model included diet as fixed effect, and room and period as random effects. For plasma AA, the average of the two weeks is presented, and the statistical analysis therefore included week as fixed effect. Initial body weight was included in the model as a covariate. Orthogonal polynomial contrast coefficients were used to determine linear and quadratic effects of increasing SID Ile:Lys on the measured parameters. The PROC NLIN and NLMIXED procedures of SAS were used to estimate the optimum Ile:Lys for ADFI, ADG, and FCR by subjecting the least square mean of the response parameters to curvilinear plateau and quadratic regression models (Robbins et al., 2006). The models are described in the figures for the response parameters. Statistical significance was accepted at P<0.05 and tendencies at P<0.10. Data are presented as least square means and SEM. 3. Results The effects of increasing the SID Ile:Lys on ADFI, ADG, and FCR as well as initial and final body weight are shown in Table 3. The ADFI increased quadratically (P<0.001) with an increasing level of the SID Ile:Lys, and the greatest ADFI was obtained

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Table 2 Analyzed and calculated composition of experimental diets with different Ile content (g/kg, as-fed)a . SIDb Ile:Lys

Item

Calculated composition Crude protein Cystine Histidine Isoleucine Leucine Lysine Methionine Methionine + Cystine Phenylalanine Threonine Valine Analyzed composition Crude protein (N × 6.25) Alanine Arginine Aspartate Cystine Glutamate Glycine Histidine Isoleucine Leucine Lysine Methionine Methionine + Cystine Phenylalanine Proline Serine Threonine Valine a b

0.42

0.46

0.50

0.54

0.58

0.62

162.8 2.7 4.4 5.4 13.7 12.2 4.5 7.2 7.6 8.2 9.5

162.8 2.7 4.4 5.9 13.7 12.2 4.5 7.2 7.6 8.2 9.5

162.8 2.7 4.4 6.3 13.7 12.2 4.5 7.2 7.6 8.2 9.5

162.8 2.7 4.4 6.8 13.7 12.2 4.5 7.2 7.6 8.2 9.5

162.8 2.7 4.4 7.2 13.7 12.2 4.5 7.2 7.6 8.2 9.5

162.8 2.7 4.4 7.7 13.7 12.2 4.5 7.2 7.6 8.2 9.5

163.9 5.6 8.2 11.0 2.7 34.4 5.7 4.4 5.6 13.3 12.2 4.3 7.0 8.5 10.5 6.9 7. 9 9.8

171.2 5.8 8.5 11.1 2.7 34.6 5.9 4.5 6.2 13.5 12.8 4.7 7.4 8.8 10.6 7.1 8.3 10.1

168.8 5.7 8.3 11.3 2.7 34.0 5.8 4.5 6.4 13.1 12.4 4.5 7.2 8.7 10.6 7.1 8.1 9.7

168.6 5.7 8.3 11.3 2.7 33.5 5.8 4.5 6.9 13.1 12.5 4.6 7.2 8.7 10.6 7.1 8.2 9.8

166.4 5.6 8.1 10.9 2.7 32.6 5.7 4.4 7.1 12.7 11.7 4.3 7.0 8.4 10.6 6. 9 7.9 9.4

168.6 5.6 8.2 11.0 2.7 32.4 5.7 4.4 7.7 12.8 12.2 4.4 7.0 8.5 10.6 7.0 7.9 9.5

Three samples of each diet were analyzed. SID = standardized ileal digestible.

Table 3 Effect of increasing standardized ileal digestible (SID) Ile:Lys on performance of the pigsa . Item

Initial BW, kg Final BW, kg ADFI d 0-14, g ADG d 0-14, g FCR d 0-14 a b c

P-valuec

SID Ile:Lys 0.42

0.46

0.50

0.54

0.58

0.62

SEMb

Linear

Quadratic

8.72 14.15 552 398 1.41

8.54 14.46 595 420 1.40

8.56 15.17 656 470 1.40

8.61 15.02 651 459 1.42

8.58 14.79 618 443 1.38

8.46 14.68 597 435 1.38

0.38 0.32 30 22 0.03

0.25 0.08 0.11 0.08 0.50

0.89 0.007 0.001 0.007 0.62

Data represents the least square means of 16 pigs per treatment. Standard error of mean. Orthogonal polynomial contrast coefficients were used to determine linear and quadratic effects of increasing standardized ileal digestible Ile to Lys.

by pigs fed the 0.50 SID Ile:Lys diet. The ADG increased quadratically (P<0.008) and tended to increase linearly (P<0.07) as the SID Ile:Lys level increased, and the highest ADG was achieved when pigs were fed the 0.50 SID Ile:Lys diet. The FCR was not affected by the dietary treatments. Final body weight increased quadratically (P<0.007) and tended to increase linearly (P<0.08) by increasing level of SID Ile:Lys in the diet, and the greatest final body weight was achieved by pigs fed the 0.50 SID Ile:Lys diet. The effect of dietary SID Ile:Lys levels on plasma urea nitrogen (PUN) and free AA are presented in Table 4. There was a linear increase in the plasma Ile concentration with an increasing level of SID Ile:Lys in the diet (P<0.001). The plasma Leu concentration showed a quadratic decrease (P<0.03) and tended to decrease linearly (P<0.10) with increasing the level of SID Ile:Lys in the diet. There were also linear decreases in plasma glycine (P<0.001) and serine (P<0.004) concentrations when the level of SID Ile:Lys in the diet increased. The concentration of the other AA in plasma showed no difference among the dietary treatments. Dietary SID Ile:Lys levels did not affect urinary urea to creatinine ratio on day 8. The urinary urea to creatinine ratio of the samples on day 15 was higher in pigs fed the 0.50 SID Ile:Lys diet in comparison with the 0.42 (P<0.03) and 0.62 (P<0.001) SID Ile:Lys diets (Table 5).

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Table 4 Effect of increasing standardized ileal digestible (SID) Ile:Lys on plasma urea nitrogen and amino acid concentrations of the pigsa . Item

P-valuec

SID Ile:Lys 0.42

b

0.46

0.50

0.54

0.58

0.62

SEM

Linear

PUNd , mmol/L 1.86 1.82 Plasma essential amino acids, mmol/L Arginine 0.12 0.14 Isoleucine 0.04 0.06 Leucine 0.17 0.16 Lysine 0.36 0.42 Methionine 0.10 0.11 Phenylalanine 0.12 0.11 0.36 0.38 Threonine 0.40 0.38 Valine Plasma non-essential amino acids, mmol/L Alanine 0.86 0.78 0.04 0.04 Asparatate 0.01 0.01 Cystine 0.32 0.33 Glutamine 1.01 0.94 Glycine Proline 0.47 0.44 0.30 0.25 Serine Tyrosine 0.16 0.18

2.00

2.20

1.74

1.57

0.45

0.50

0.16

0.13 0.07 0.14 0.36 0.10 0.11 0.33 0.37

0.13 0.08 0.14 0.39 0.11 0.11 0.36 0.39

0.13 0.10 0.15 0.41 0.10 0.13 0.37 0.38

0.12 0.12 0.15 0.40 0.10 0.11 0.34 0.37

0.01 0.01 0.01 0.04 0.01 0.01 0.03 0.02

0.83 <0.001 0.10 0.31 0.75 0.93 0.64 0.30

0.26 0.48 0.03 0.98 0.84 0.31 0.92 0.50

0.72 0.04 0.01 0.34 0.78 0.44 0.25 0.16

0.73 0.04 0.01 0.35 0.85 0.45 0.23 0.18

0.85 0.04 0.01 0.36 0.85 0.46 0.24 0.19

0.76 0.04 0.01 0.34 0.77 0.42 0.23 0.18

0.07 0.002 0.002 0.03 0.08 0.03 0.02 0.02

0.51 0.33 0.43 0.30 <0.001 0.35 0.003 0.15

0.13 0.66 0.06 0.62 0.13 0.78 0.11 0.77

a b c d

Quadratic

Data represents the least square means of plasma samples from 8 pigs per treatment. Standard error of mean. Orthogonal polynomial contrast coefficients were used to determine linear and quadratic effects of increasing standardized ileal digestible Ile to Lys. PUN = plasma urea nitrogen.

Table 5 Effect of increasing standardized ileal digestible (SID) Ile:Lys on urine urea to creatinine ratio (U:C) of the pigsa . Item

U:C, d8 U:C, d15 a b c

P-valuec

SID Ile:Lys 0.42

0.46

0.50

0.54

0.58

0.62

SEMb

Linear

Quadratic

22.14 32.87

15.06 38.22

19.77 49.70

18.91 41.12

18.83 36.24

20.24 31.11

7.26 7.25

0.97 0.59

0.38 0.02

Data represents the least square means of urine samples from 8 pigs per treatment. Standard error of mean. Orthogonal polynomial contrast coefficients were used to determine linear and quadratic effects of increasing standardized ileal digestible Ile to Lys. 700

Average daily feed intake, g/day

680 660 640 620 600 580 560 540 520 500 0.40

0.44

0.48

0.52

0.56

0.60

0.64

Dietary SID Ile to Lys ratio Fig. 1. The optimum SID Ile:Lys determined by a curvilinear-plateau model was 0.51 (Y = 626–7481 (0.51 − x)2 ) (solid line). The data was also fitted to a quadratic regression model (Y = −8006.5x2 + 8442.6x − 1576.3) (dashed-line). The upper asymptote of the quadratic function was calculated to be 0.53 SID Ile:Lys. The 95% confidence limits ranged from 0.42 to 0.60 and from 0.49 to 0.58 for the curvilinear plateau and quadratic regression models, respectively. The quadratic regression model fitted the data slightly better according to the Akaike Information Criterion (AIC). Data points (•) represent mean ± SEM for each dietary treatment (n = 16 pigs per treatment).

Animal performance traits were assessed by different models to estimate the minimum SID Ile:Lys that supports the maximum animal response and the best fitting models were chosen according to the Akaike Information Criterion (AIC) (Akaike, 1978). The curvilinear plateau and quadratic regression models were chosen to describe the Ile requirement for ADFI and ADG, and the broken-line model was chosen for FCR, respectively (Figs. 1, 2 and 3). Considering quadratic regression models to describe the requirements, the maximum performance was obtained at 0.53 and 0.53 SID Ile:Lys for ADFI and ADG, respectively. The 95% confidence limits ranged from 0.49 to 0.58 and from 0.48 to

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Average daily gain, g/day

490 470 450 430 410 390 370 350

0.40

0.44

0.48

0.52

0.56

0.60

0.64

Dietary SID Ile to Lys ratio Fig. 2. The optimum SID Ile:Lys determined by a curvilinear-plateau model was 0.52 (Y = 448–4718 (0.52 − x)2 ) (solid-line). The data were also fitted to a quadratic regression model (Y = −4792.2x2 + 5101x − 896.89) (dashed-line). The upper asymptote of the quadratic function was calculated to be 0.53 SID Ile:Lys. The 95% confidence limits ranged from 0.43 to 0.61 and from 0.48 to 0.58 for curvilinear plateau and quadratic regression models, respectively. The quadratic regression model fitted the data slightly better according to the Akaike Information Criterion (AIC). Data points (•) represent mean ± SEM for each dietary treatment (n = 16 pigs per treatment). 1.50

Feed conversion ratio

1.48 1.46 1.44 1.42 1.40 1.38 1.36 1.34 1.32 1.30

0.40

0.45

0.50

0.55

0.60

0.65

Dietary SID Ile to Lys ratio Fig. 3. The optimum SID Ile:Lys determined by a broken-line model was 0.48 (Y = 1.39 + 0.65 (0.48 − x), 95% confidence limits ranged from 0.42 to 0.54. Data points (•) represent mean ± SEM for each dietary treatment (n = 16 pigs per treatment).

0.58 SID Ile:Lys for ADFI and ADG, respectively. Curvilinear plateau models estimated the maximum ADFI and ADG at 0.51 and 0.52 SID Ile:Lys, respectively. The 0.95 confidence limits ranged from 0.42 to 0.60 and from 0.43 to 0.61 SID Ile:Lys for ADFI and ADG, respectively. Considering FCR as the response, only a broken-line model could be fitted to the data, and 0.48 SID Ile:Lys was estimated as the requirement with confidence limits ranging from 0.42 to 0.54 SID Ile:Lys. 4. Discussion Following the ideal protein concept, the requirement of individual AA is defined as the minimum level of the AA in ratio to lysine, which optimizes growth, nitrogen retention, or another response criterion (Boisen, 1997). Lysine is a reference for the other indispensable AA because it is commonly the first limiting AA in practical pig diets, and its requirement has been well investigated (Boisen, 2003). To get an empirical approach of Ile requirement, Lys should be the second limiting AA in a dose-response study (Boisen, 2003). Therefore, diets were formulated to be sub-limiting in Lys (0.93 of requirements). The mean analyzed Lys content was 12.3 g/100 g feed and was not different from the expected value and among dietary treatments. There are only few studies that estimate the Ile requirement in cereal-based diets without using blood cells (Barea et al., 2009; Nørgaard et al., 2013). A meta-analysis on literature on Ile requirement was carried out by Van Milgen et al. (2012). From a total of 46 experiments on Ile dose-response, 33 reported a response to increasing level of Ile in the diet. One of the main factors of variation among the experiments was the inclusion of blood products in the diet. The blood products contain low concentration of Ile, but are rich in Leu, Val, His, and Phe (Van Milgen et al., 2012). The competition of BCAA for catabolic enzymes and transportation causes an increase in the requirement of Ile, when the diet contains the Leu, Val, His, and Phe rich blood products. Therefore, the requirements of SID Ile:Lys was reported as 0.50 and 0.55 for pigs fed diets without or with blood cells, respectively. In the current study, the estimated SID Ile:Lys requirements was 0.48 to 0.53 depending on the chosen response parameter and statistical model, which is in agreement with the estimates by Van Milgen et al. (2012).

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The results of the current study are also consistent with the work of Wiltafsky et al. (2009) who evaluated Ile requirements in ratio to Lys in diets both with and without blood cells. These authors estimated the requirements of SID Ile:Lys to be 0.54, 0.54, and 0.49 for ADG, ADFI, and G:F, respectively, in cereal-based diets without blood cells and 0.59 SID Ile:Lys for ADFI and ADG in diets containing blood cells. A recent study by Nørgaard et al. (2013) estimated the optimum level of SID Ile:Lys at 0.52, 0.52, and 0.52 for ADFI, ADG, and G:F, respectively using a quadratic regression model, and 0.50, 0.53, and 0.54 SID Ile:Lys for maximum ADFI, ADG, and G:F, respectively using quadratic broken-line model. In the current study, the estimated requirement also showed minor discrepancies according to the chosen statistical model. The estimated requirements for maximum ADFI were 0.51 and 0.53 SID Ile:Lys by curvilinear plateau model and quadratic regression model, respectively. Similar small difference in optimum SID Ile:Lys for maximum ADG was also observed (0.52 vs. 0.53 in curvilinear plateau and quadratic regression models, respectively). As there was a decline in ADFI and ADG after maximum performance, the quadratic regression model fitted the data better than curvilinear plateau model which was reflected in slightly smaller AIC and 95% confidence limits in quadratic regression model than curvilinear plateau model (data are not presented). In the FCR data, alternative models were evaluated but according to the modality of the FCR data, only a broken-line model fitted the data and 0.48 SID Ile:Lys was estimated as the requirement for the lowest FCR. The lower estimate of requirements using FCR as the response is partly due to the fitted broken-line model to the data which often forces the breakpoint to a lower requirement (Robbins et al., 2006). Also as described in Table 3 there is no consistent response to FCR. Thus, the results of the current study are in agreement with the recent studies on SID Ile:Lys requirements using blood free diets and suggesting a range between 0.50 and 0.54 SID Ile:Lys for the optimum animal performance (Wiltafsky et al., 2009; Gloaguen et al., 2013; Nørgaard et al., 2013). It should be noticed that the upper 95% confidence limits in the current study were up to 0.61 SID Ile:Lys. These rather large confidence limits may argue for higher Ile concentrations in practical diets if growth retardation is observed. In the current study, there was a decline in ADFI and ADG after the optimum SID Ile:Lys. The impaired animal performance at high Ile intake, which is also reported by Nørgaard et al. (2013) and Wiltafsky et al. (2009), could be derived from the interaction among BCAA. As it has been shown previously (Wiltafsky et al., 2010), excess Leu causes more degradation of Ile. This is due to the increased ␣-keto-isocaproate (KIC) formed from transamination of Leu which stimulates the branchedchain keto acid dehydrogenase complex (BCKDH). The BCKDH catalyzes an irreversible step of all BCAA catabolism. It seems that ␣-keto-␤-methylvalerate (KMV) produced from excess Ile transamination may stimulate the enzyme in a similar way, and therefore, by increasing the SID Ile:Lys probably, Leu becomes limiting. The response curve showed that Ile supply 10% below or above the requirements causes 3% decrease in ADFI, respectively. The slope for ADG curve also showed that Ile supply 10% below or above the requirements results in 3% reduction in animal growth response. The response to Ile deficiency was also reported in meta-analyses done by Van Milgen et al. (2012) who reported 15% reduction in ADFI and 21% reduction in ADG of the pigs when fed diets 10% deficient in Ile. Increasing the level of SID Ile:Lys in the diet caused a linear increase in the plasma Ile concentration (P<0.001), which was along with a quadratic decrease in Leu (P<0.03) concentration and linear decrease in glycine (P<0.001) and serine (P<0.004) concentrations in the plasma of the pigs. Similar decrease in plasma glycine and serine concentration by increased dietary SID Ile:Lys were reported by Wiltafsky et al. (2009). Because urea is one of the main AA transamination products of AA catabolism, PUN (Pedersen and Boisen, 2001; Parr et al., 2003) or urinary urea nitrogen (Brown and Cline, 1974) could be a reflection of a balanced diet regarding the AA profile. It is believed that AA imbalance would result in the transamination of all the AA which will increase the urea content in the blood and/or urine. In our study, however, neither PUN nor urinary urea nitrogen showed any special trend at the different SID Ile:Lys, which implies that these parameters could not be used as indicators of an imbalanced AA profile in the present experiment. 5. Conclusions The optimum SID Ile:Lys for the maximum animal performance was obtained from 0.51 to 0.53 SID Ile:Lys depending on the chosen performance trait and/or statistical model. From these values, 0.52 SID Ile:Lys could be concluded as the requirement. The plasma concentration of BCAA confirms the interaction among these AA which could be due to the shared enzymes in their catabolic pathways. Neither PUN nor urinary urea nitrogen showed a clear relation to the Ile:Lys level or animal performance and therefore could not be used as an indication of AA imbalance in the current study. Conflicts of interest The authors have no conflict of interest. Acknowledgements The authors greatly acknowledge the financial support by Ajinomoto Eurolysine S.A.S., The Danish Council for Independent Research – Technology and Production Science, and Aarhus University.

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