presentación metabolismo

N. Litvak, A. Rakhshandeh, J. K. Htoo and C. F. M. de Langeplus cysteine ratio in growing pigs

Immune system stimulation increases the optimal dietary methionine to methionine

doi: 10.2527/jas.2012-6160 originally published online July 3, 20132013, 91:4188-4196.J ANIM SCI

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Immune system stimulation increases the optimal dietary methionine to methionine plus cysteine ratio in growing pigs1

N. Litvak,* A. Rakhshandeh,† J. K. Htoo,‡ and C. F. M. de Lange*2

*Department of Animal and Poultry Science, University of Guelph, Guelph, ON N1G 2W1, Canada; †Department of Animal Science, Iowa State University, Ames 50011; and ‡Evonik Industries AG, Hanau 63457, Germany

ABSTRACT: Chronic subclinical levels of disease occur frequently in intensive swine production and compromise nutrient use efficiency. Feeding additional Met plus Cys (M+C) has been implicated in improving the response of the animal to immune system stimulation (ISS) because they can serve as substrates for generating compounds involved in the immune response, such as glutathione and acute phase proteins. A N-balance study was conducted to assess the optimal dietary Met to Met plus Cys ratio (M:M+C) during ISS in 20-kg pigs. Thirty-six pigs were fed 800 g/d of 1 of 5 M+C-limiting diets, containing graded levels of M:M+C (0.42, 0.47, 0.52, 0.57, and 0.62) and supplying 2.5 g/d of M+C. After adaptation, N bal-ances were determined sequentially during a 5-d prechal-lenge period and 2 ISS periods of 3 and 4 d, respectively. To induce ISS, pigs were injected intramuscularly with

repeated and increasing doses of Escherichia coli lipo-polysaccharide. Eye temperature and blood profile con-firmed effective ISS. During ISS period 1, ISS reduced the mean N balance more severely than ISS period 2 (8.7 ± 0.3 vs. 9.6 ± 0.4 g/d; P < 0.001) and was less than the pre-challenge period (10.0 ± 0.2 g/d; P < 0.001). An interac-tive effect of ISS and diet on N balance was observed (P < 0.001). Based on quadratic-plateau regression analysis, the optimal dietary M:M+C was 0.57 ± 0.03 and 0.59 ± 0.02 for the prechallenge period and ISS period 2, respec-tively. The optimal dietary M:M+C for ISS period 1 was found to be greater than 0.62, indicating that the optimal M:M+C is greater during ISS. It is suggested that this is the result of preferential use of Met during ISS. In con-clusion, ISS results in an increase in the optimal dietary M:M+C in growing pigs.

Key words: body protein deposition, cysteine, immune system stimulation, inflammation, methionine, pigs

© 2013 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2013.91:4188–4196 doi:10.2527/jas2012-6160

INTRODUCTION

Whole body protein deposition (Pd) represents the balance between the concurrent processes of body protein synthesis and body protein degradation (Metayer et al., 2008). In an effort to optimize Pd and nutrient utilization in growing pigs, there is a need to determine the dietary AA requirements for Pd at the various stages of production and under varying environmental conditions. The AA requirements of growing pigs have been studied extensively (NRC, 2012). However, there is limited information about

the impact of immune system stimulation (ISS; e.g., disease) on AA requirements of growing pigs even though it has been shown that some AA have important roles in the immune response of the animal (Hunter and Grimble, 1994, 1997; Malmezat et al., 2000b; Melchior et al., 2004).

It has been noted that during ISS, Cys is released from skeletal muscle protein in quantities closest to the theoretical requirements for the synthesis of acute phase proteins (APP; Reeds et al., 1994) and that ISS leads to enhanced Cys utilization for the synthesis of compounds involved in the immune response, such as glutathione (GSH; Malmezat et al., 2000a). These findings indicate that Cys is limiting for supporting the immune response, unless it is adequately supplemented in the diet. Because Met can be converted to Cys through the transsulfuration (TS) pathway but not vice versa (Lu, 2009), it has been suggested that there is a minimum dietary Met to Met plus Cys

1Supported by funds from Ontario Ministry of Agriculture, Food and Rural Affairs (Guelph, ON, Canada), Ontario Pork (Guelph, ON, Canada), Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada), and Evonik Industries (Hanau, Germany).

2Corresponding author: [email protected] December 6, 2012.Accepted May 30, 2013.



Methionine and cysteine ratio for growing pigs 4189

(M+C) ratio (M:M+C), at which M+C utilization for Pd is maximized. The present study was undertaken to determine the optimal M:M+C during ISS in growing pigs using the N-balance method.

MATERIALS AND METHODS

All procedures in this study were conducted in accordance with the Canadian Council on Animal Care (CCAC, 2009) and approved by the University of Guelph Animal Care Committee.

Animals, Housing, Diets, and General Experimental Design

Thirty-six Yorkshire barrows were selected from the University of Guelph Arkell Swine research facility herd and housed individually in floor pens at the University of Guelph Animal Metabolism Unit (Möhn et al., 2000). At an initial BW of 17.3 ± 2.0 kg, pigs were assigned to 1 of 5 dietary treatments that represented varying M:M+C and were adjusted to dietary treatments during a 6-d period. On d 4 of the adjustment period and 2 d before the start of N-balance observations, pigs were moved from the floor pens to metabolism crates (Möhn et al., 2000). In all pigs, whole body N balances were measured during a 5-d period before ISS (prechallenge period). At the completion of the prechallenge period, ISS was induced using repeated intramuscular injections of Escherichia coli lipopolysaccharide (LPS; strain 055:B5; Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) every 48 h for 7 d. The initial dose was 50 µg of LPS/kg BW on d 1 of ISS, and this dose was increased by 12% at each injection to overcome potential tolerance to LPS, with an increase of 15% on the last day of the 7 d ISS period (Rakhshandeh and de Lange, 2012).

After initiation of ISS, N balances were measured during 2 consecutive periods of 3 and 4 d, respectively, for ISS periods 1 and 2 to assess the time dependent effect of ISS on N use. Pigs were weighed at the start and end of the prechallenge period and again after the completion of ISS period 2. The experiment was conducted in 3 blocks consisting of 12 pigs each. In each block, 2 or 3 pigs were assigned to each of the dietary treatments. Full details of the N-balance procedures are provided by Möhn and de Lange (1998). The only alteration to these procedures was the omission of urinary catheters for the collection of urine; urine was collected directly via a urine collection tray that drained into collection containers, which contained sufficient HCl to reduce pH below 3 to avoid N losses by volatilization of ammonia (de Lange et al., 2001).

The experimental diets were formulated based on energy and nutrient contents in ingredients accord-ing to NRC (1998) to be isonitrogenous, isoenergetic

(3,895 kcal ME/kg DM), and first limiting in M+C (Gil-lis et al., 2007), providing approximately 2.5 g/d of total M+C intake at the targeted feed intake of 800 g/d (Ta-ble 1). On a molar basis, the 5 diets contained the same amount of total M+C from casein and crystalline AA. On a weight basis, the target total M:M+C were 0.42, 0.47, 0.52, 0.57, and 0.62, respectively. The different M:M+C in the casein and cornstarch-based diets were generated by varying the amounts of added crystalline Met and Cys. Diets 1 and 5 were prepared in single batches at the University of Guelph Arkell Feed Mill whereas diets 2, 3, and 4 were produced by blending diets 1 and 5 in varying proportions to achieve the target M:M+C. The

Table 1. Ingredient composition (%) of the 2 experimental diets with the extreme ratios of methionine to methionine plus cysteine (M:M+C)1

Ingredient

Dietary M:M+C0.42 0.62

Cornstarch 48.64 48.71Sucrose 20.00 20.00Sodium caseinate 4.70 4.70Cellulose 4.00 4.00Corn oil 4.00 4.00Limestone 0.80 0.80Dicalcium phosphate 3.00 3.00Salt 0.40 0.40Potassium chloride 0.90 0.90Magnesium sulfate 0.35 0.35Vitamin–mineral mix2 0.50 0.50Titanium dioxide 0.10 0.10dl-Met 0.00 0.07l-Cys HCl 0.23 0.15l-Lys HCl 0.49 0.49l-Thr 0.35 0.35l-Trp 0.12 0.12l-Ile 0.29 0.29l-Val 0.29 0.29l-His 0.15 0.15l-Phe 0.46 0.46l-Leu 0.46 0.46l-Asp 4.83 4.83l-Glu 4.88 4.88Choline chloride 0.06 0.06

1The experimental diets with intermediate M:M+C (0.47, 0.52, and 0.57) were prepared by blending the 0.42 and 0.62 M:M+C diets in the appropriate proportions: 75% of the 0.42 M:M+C diet and 25% of the 0.62 M:M+C diet, 50% of the 0.42 M:M+C diet and 50% of the 0.62 M:M+C diet, and 25% of the 0.42 M:M+C diet and 75% of the 0.62 M:M+C diet, respectively.

2Supplied per kilogram of complete diet: vitamin A, 10,000 IU as retinyl acetate (2.5 mg) and retinylpalmitate (1.7 mg); vitamin D3, 1,000 IU as cholecalciferol; vitamin E, 56 IU as dl-α-tocopherol acetate (44 mg); vitamin K, 2.5 mg as menadione; choline, 500 mg; pantothenic acid, 15 mg; riboflavin, 5 mg; folic acid, 2 mg; niacin, 25 mg; thiamine, 1.5 mg; vitamin B6, 1.5 mg; biotin, 0.2 mg; vitamin B12, 0.025 mg; Se, 0.3 mg from Na2SeO3; Cu, 15 mg from CuSO4.5H2O; Zn, 104 mg from ZnO; Fe, 100 mg from FeSO4; Mn, 19 mg from MnO2; and I, 0.3 mg from KI (DSM Nutritional Products Canada Inc., Ayr, ON, Canada).



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diets contained TiO2 (Sigma-Aldrich Canada Ltd.) as an indigestible marker for determining nutrient digestibility and fecal nutrient excretion. All diets were supplement-ed with vitamins and minerals to exceed requirements according to NRC (1998).

As ISS has been shown to reduce voluntary feed intake (Williams et al., 1997), animals were feed restricted to eliminate potential confounding of feeding level with ISS, and feed intake was maintained constant across all diets and experimental periods, based on previous feed intake data from our laboratory (Rakhshandeh and de Lange, 2012). Pigs were fed 2 equal meals daily at 0800 and 1600 h.

Measurement of Eye Temperature and Blood Sampling

An infrared camera (FLIR Technologies, Burlington, ON, Canada) was used to determine the eye temperature of each pig just before injection with LPS and at 2 and 6 h and every 24 h after the first injection. As eye temperature is correlated to core body temperature (Kessel et al., 2010) and is less invasive than measurement of rectal temperature, this measurement was used to indicate potential fever and, thus, effectiveness of ISS.

Just before the first injection with LPS and again at the completion of the ISS period, 3 blood samples of 5 mL each were collected at 1000 h from the orbital sinus from one-half of the pigs within each treatment and stored in blood collection tubes that contained either EDTA, heparin, or buffered sodium citrate (BD Vacutainers, Mississauga, ON, Canada). Blood samples in tubes containing EDTA were submitted immediately for the measurement of white blood cell count. Heparinized and buffered sodium citrate blood samples were kept on ice until centrifugation at 3,000 × g at 4°C for 20 min. The plasma was then isolated and submitted immediately for analysis of haptoglobin, albumin, and fibrinogen concentration, respectively.

Analytical Procedures

All blood samples were analyzed at Animal Health Laboratories (University of Guelph, Guelph, ON, Canada). White blood cell count was determined using cytogram analysis (Advia 120 Hematology System; Siemens Healthcare Diagnostics IN., Deerfield, IL). Plasma haptoglobin and albumin were measured (Roche Cobas C 501 Analyzer; Hoffman-La Roche Ltd., Mississauga, ON, Canada) according to methods described by Makimura and Suzuki (1982) and Doumas et al. (1971), respectively. Plasma fibrinogen was measured using a kit (TriniCLOT Fibrinogen; catalog number T1301) and based on coagulation (KC4 Delta Semi-Automatic Coagulation Analyzer; Trinity Biotech Plc., Jamestown, NY).

Diet samples were analyzed for AA composition using ion-exchange chromatography with post-column derivatization with ninhydrin (Evonik Industries AG, Hanau, Germany; Llames and Fontaine, 1994). Dry matter content of the diets, feces, and wasted feed was measured in duplicate by oven drying for 2 h at 135°C (Method 930.15; AOAC, 1990). Diet and fecal samples were analyzed for TiO2 content in triplicate and duplicate, respectively (AOAC Int., 1997). Nitrogen content was quantified in triplicate for diet samples and in duplicate for fecal and urine samples using an automatic analyzer (LECO-FP 428; Leco Instruments Ltd., Mississauga, ON, Canada; Method 990.03; AOAC Int., 1997).

Calculations and Statistical Analysis

Total tract DM digestibility and fecal N excretion were determined using the indicator technique (Zhu et al., 2005). The mean measured dietary Ti content was used in the calculations of fecal N digestibility. Nitrogen retention (g/d) was calculated as net N intake (feed N – feed wastage N) minus N excretion (fecal N plus urinary N). Protein deposition (g/d) was calculated as retained N × 6.25.

Statistical analyses were performed using the mixed model procedures (SAS Inst. Inc., Cary, NC). The eye temperature and plasma APP concentrations were analyzed as repeated measures on pigs with diet (n = 5; fixed effect), period (n = 2; fixed effect), and pigs within block and diet included in the model as random effects. Because of the interactive effect of dietary treatment and N-balance period (P < 0.05), dietary treatment effects on the N-balance data were analyzed separately for each N-balance period, with diet (n = 5; fixed effect) and block (n = 3; random effect) included in the model as sources of variation. In these models, block effects were not significant (P > 0.10). Orthogonal polynomial contrasts were performed for each N-balance period to determine linear and quadratic effects of dietary M:M+C. Linear- and quadratic-plateau models were evaluated, using the NLIN procedure of SAS, with significance accepted at P < 0.05. Probabilities between 0.05 and 0.10 were considered to indicate a trend.

RESULTS

General ObservationsBefore ISS, pigs appeared healthy and readily

consumed their daily feed allowances. The first LPS injection induced vomiting in all pigs. Vomitus was collected and included in wasted feed; however, its contribution to wasted feed DM was minimal. The data from 2 pigs were excluded from the study; 1 pig behaved abnormally in the metabolism crate, resulting




in incomplete urine collection, and the other suffered from diarrhea, resulting in contamination of urine with feces. In addition, some N-balance observations were missing due to incomplete collection of urine or insufficient collection of feces. In total, there were 4, 5, and 3 missing observations in the prechallenge, ISS period 1, and ISS period 2, respectively.

Calculated and analyzed dietary nutrient contents are presented in Table 2. For most AA, the analyzed AA contents were similar across diets and in agreement with calculated values based on the 1998 NRC ingredient compositions. For Met and Cys, analyzed AA contents were very similar to calculated values. The slight discrepancies between analyzed and calculated values may be attributed to lack of repeatability in AA analyses (Rutherford and Moughan, 2000) or inaccuracies in diet preparation. Given that extreme care was taken in diet manufacturing [i.e., use of well-defined and well-characterized dietary AA sources, blending the 2 extreme diets (diets 0.42 and 0.62 M:M+C) to prepare the intermediate diets (diets 0.47, 0.52, and 0.57 M:M+C)], calculated rather than analyzed N and AA contents were used in the interpretation of the results. The calculated M+C to Lys ratio in the diets varied between 0.41 and 0.43 whereas the ratios of the other indispensable AA exceeded the minimum ratios according to NRC (1998, 2012), indicating that M+C was first limiting AA in all diets.

There was no interactive effect of diet and N-balance period on any of the blood profile (Table 3). The N-balance period effect for blood haptoglobin, albumin, and fibrinogen concentrations was large (P < 0.001). Both haptoglobin and fibrinogen concentrations increased during ISS whereas albumin concentrations were reduced. There was a dietary treatment effect on the plasma concentration of albumin (P = 0.01). This difference is attributed to the greater plasma albumin

concentration in pigs consuming the 0.42 M:M+C diet before the ISS period (P < 0.05; Table 4).

There were no block and diet effects or interactive effect of time and diet on eye temperature (data not shown) whereas there was a time effect (P < 0.001). Eye temperature was greater at 2, 6, 48, 96, and 144 h after the start of ISS compared with the prechallenge value (time 0; P < 0.02), confirming effective ISS.

Nitrogen Balance

Data on aspects of N utilization and Pd during the prechallenge and 2 ISS periods are shown in Table 5. In all periods, BW and apparent fecal CP digestibility were not affected by dietary treatment. Mean BW were 18.0 ± 1.0, 21.6 ± 1.0, and 23.5 ± 0.9 kg whereas CP digestibility was 92.9 ± 0.8, 91.8 ± 0.8, and 92.8 ± 0.6% for the prechallenge and ISS periods 1 and 2, respectively.

During the prechallenge period, urinary N excretion was highly affected by dietary treatments (P = 0.002). The marginal reduction in urinary N excretion with increasing M:M+C was smaller at the greatest M:M+C (linear and quadratic, P < 0.02). A dietary effect was observed for Pd in the prechallenge period (P < 0.001). The Pd increased with increasing dietary M:M+C and reached a plateau at the greater dietary M:M+C (linear and quadratic, P < 0.001).

Although LPS injection induced temporary anorexia in pigs, this was usually overcome within 12 h of injection. Pigs consumed their daily feed allowance on days of LPS injection, with the exception of pigs consuming the 0.42 M:M+C diet. As a result, N intake in ISS period 1 differed among dietary treatments (P < 0.001). Yet Pd (or N retention) was different among dietary treatments and Pd increased with increasing dietary M:M+C (linear, P <

Table 2. Calculated and analyzed nutrient contents (%, as-is basis) in the experimental diets1,2

Item

Dietary M:M+C0.42 0.47 0.52 0.57 0.62

Anal Calc Anal Calc Anal Calc Anal Calc Anal CalcCP 11.9 11.2 13.3 11.2 12.9 11.2 12.5 11.2 12.4 11.2Met 0.12 0.13 0.15 0.14 0.17 0.16 0.18 0.18 0.18 0.19Cys 0.16 0.17 0.15 0.16 0.14 0.15 0.13 0.13 0.12 0.12Met + Cys 0.27 0.30 0.31 0.30 0.31 0.31 0.31 0.31 0.31 0.31Lys 0.77 0.73 0.76 0.73 0.77 0.73 0.84 0.73 0.75 0.73Thr 0.47 0.53 0.52 0.53 0.56 0.53 0.50 0.53 0.52 0.53Trp 0.12 0.17 0.14 0.17 0.16 0.17 0.15 0.17 0.15 0.17Ile 0.46 0.51 0.52 0.51 0.49 0.51 0.49 0.51 0.47 0.51Leu 0.83 0.87 0.91 0.87 0.91 0.87 0.88 0.87 0.87 0.87Val 0.55 0.58 0.59 0.58 0.56 0.58 0.57 0.58 0.55 0.58His 0.25 0.28 0.28 0.28 0.29 0.28 0.27 0.28 0.27 0.28Phe 0.58 0.68 0.63 0.68 0.67 0.68 0.64 0.68 0.65 0.68

1Calculated values represent contributions from crystalline AA and the AA composition of casein according to NRC (1998).2M:M+C = Met to Met plus Cys ratio; Anal = analyzed values; and Calc = calculated values.




0.001). Similarly, in ISS period 2, linear and quadratic increases in Pd (P < 0.03) were observed with increasing M:M+C, reducing the increase in Pd with increasing M:M+C at the greater dietary M:M+C.

An interactive effect of dietary treatment and N-balance period on Pd was observed (P < 0.001). Protein deposition differed among periods only for the diets containing 0.47, 0.52, and 0.62 M:M+C. For these 3 dietary treatments, Pd was less in ISS period 1 than in the prechallenge period (P < 0.05) whereas Pd in ISS period 2 did not differ from either the prechallenge period or ISS period 1. A similar trend towards a period effect on Pd was observed for the diets containing 0.42 (P = 0.06) and 0.57 (P = 0.09) M:M+C.

Both linear- and quadratic-plateau models were evaluated for determining a breakpoint corresponding to the M:M+C for maximizing Pd in each period (Fig. 1). The quadratic-plateau regression analysis yielded better fits to the data with R2 of 0.98, 0.98, and 0.99 for the prechallenge and ISS periods 1 and 2, respectively. Based on the quadratic-plateau model, the breakpoints were estimated to be 0.57 ± 0.03 (Fig. 1A) and 0.59 ± 0.02 M:M+C (Fig. 1C), for the prechallenge period and ISS period 2, respectively. There was only a linear effect for ISS period 1, indicating that the optimal M:M+C requirement during this period is greater than 0.62 (Fig. 1B).

DISCUSSION

The main objective of the current experiment was to determine the effect of ISS on the optimal M:M+C in diets for growing pigs. Repeated administration of increasing doses of LPS was selected as the model for noninfectious immune challenge, largely based on previous work in our laboratory (Rakhshandeh and de Lange, 2012). The LPS model, which has been used widely, results in a predictable immune response and is possibly more representative of a disease challenge than exposing pigs to an individual infectious pathogen (Boosman et al., 1989; Dritz et al., 1996; Barnes et al., 2002; Melchior et al., 2004). In the current study, observed changes in eye

temperature and plasma concentrations of APP confirmed that ISS was achieved. The observation that greater plasma albumin concentrations were observed in pigs on the lowest dietary M:M+C is difficult to interpret. It may be speculated that increased hepatic albumin synthesis reflects increased need for endogenous Met to support the immune response and that albumin is used to transfer Met. A key concern with the LPS model is the development of tolerance (Deitch, 1998; Rakhshandeh and de Lange, 2012). To overcome tolerance, increasing amounts of LPS were injected in the current study. However, based on changes in eye temperature and Pd over time, the pigs in the current experiment did develop some tolerance to LPS. It should be noted, though, that plasma concentrations of APP may remain increased for 4 to 7 d after ISS (Petersen et al., 2004). Therefore, the observed plasma concentrations of APP provide no insight on the potential development of tolerance to the repeated LPS injections over time. To account for the development of tolerance to LPS, N-balance data during ISS are analyzed separately for the 2 consecutive N-balance periods.

In a previous study, which was conducted to explore the impact of ISS on N use in pigs fed varying levels of M+C, it was clearly shown that Pd was independent of BW in non-ISS pigs between 20 and 40 kg BW (Rakhshandeh et al., 2007) whereas the effect of BW on the optimum M:M+C is minimal (NRC, 2012). Therefore, the observed reduction in Pd during ISS period 1 can be attributed to ISS. Moreover, in the present study, pig performance was sensitive to M+C intake, as care was taken to formulate diets that contained equal molar amounts of M+C and varied only in the ratio of M:M+C. The calculated M+C to Lys ratio in the diets varied between 0.41 and 0.43 whereas the ratios of the other indispensable AA exceeded the minimum ratios according to NRC (1998, 2012), indicating that either Met or M+C was the first limiting AA, including total protein, in all diets. Also, in a previous study in our laboratory, it was shown that either Met or M+C was first limiting AA in growing pigs fed AA profiles similar to those used in the current study (Gillis et

Table 3. Plasma concentrations of acute phase proteins and white blood cell count before (prechallenge) and during immune system stimulation (ISS)1,2

Item

Period3 P-valuePrechallenge ISS Diet Period Diet × period

WBC4 count (× 109/L) 22.4 ± 1.6 22.0 ± 0.7 0.46 0.82 0.96Haptoglobin, g/L 0.41 ± 0.07 0.90 ± 0.07 0.68 <0.001 0.70Albumin, g/L 34.0 ± 0.5 29.8 ± 0.5 0.01 <0.001 0.67Fibrinogen, g/L 1.58 ± 0.11 2.48 ± 0.10 0.70 <0.001 0.40

1Immune system stimulation (ISS) was induced with increasing doses of Escherichia coli lipopolysaccharide on d 1, 3, 5, and 7 of the ISS period.2The data presented are means ± SE (n = 15 to 17 pigs) and based on blood samples taken 1 d before ISS and d 8 after the start of ISS.3Periods represent d 5 before ISS (prechallenge) and d 7 after start of ISS.4WBC = white blood cell.




al., 2007). Finally, Pd was less than typical values for this population of pigs (Möhn and de Lange, 1998), indicating that nutrient intake, rather than energy intake or the performance potentials of the pigs, determined Pd.

In the current experiment, the response to M:M+C was evaluated when pigs were healthy (before ISS) and when they were exposed to a noninfectious immune challenge (during ISS). However, because of the interactive effect of N-balance period and dietary treatment and the confounding of pigs with M:M+C, the response of pigs to M:M+C was evaluated separately for the 3 consecutive N-balance periods. Several models were fitted to the data to examine the response of Pd to varying M:M+C. It was found that, based on R2 values, the quadratic-plateau model fitted the data better than the linear-plateau model. This is consistent with previous studies, in which the response of groups of animals to varying AA intakes has been explored (Curnow, 1973; Rodehutscord and Pack, 1999; Baker et al., 2002; Heger et al., 2008).

During the prechallenge N-balance period, there was no effect of dietary M:M+C on total N intake whereas urinary N excretion decreased with increasing M:M+C. Urinary N excretion reflects both minimum plus inevitable AA catabolism and catabolism of AA that are supplied in excess of requirements for Pd (NRC, 2012), which is determined by the supply of the first limiting dietary AA, which was either Met or M+C in the current study. Based on the quadratic-plateau regression analysis, the optimal M:M+C during the prechallenge period was determined to be 0.57. This is in close agreement with the value established previously for the same population of pigs (Gillis et al., 2007; 0.55) and somewhat greater than a recent review of the literature (0.53 for 11- to 25-kg pigs; NRC, 2012).

In the current study and when the pigs were challenged with LPS, M+C was diverted from Pd

and towards supporting the immune system, which is consistent with previous observations (Husband, 1995; Colditz, 2004; Le Floc’h et al., 2004). Apparently, during ISS, M+C, and Cys in particular, are directed towards production of compounds that are involved in the immune response, such as GSH and APP (Reeds et al., 1994; Malmezat et al., 1998, 2000a,b; Grimble, 2002; Metayer et al., 2008).

Although previous studies have implied an increase in Cys needs relative to Met during ISS (Grimble, 1992; Hunter and Grimble, 1994, 1997; Jahoor et al., 1995; Malmezat et al., 1998, 2000a,b; Breuille et al., 2006), the results of the current study indicated that the optimal M:M+C is increased during ISS. The latter indicates an increased dietary requirement for Met relative to Cys during ISS. There are several potential explanations for the current finding. First, previous research examined varying levels of M+C intake and not M:M+C per se (Hunter and Grimble, 1994, 1997; Malmezat et al., 2000b; Breuille et al., 2006; Rakhshandeh et al., 2007, 2010a,b,c). This is in contrast with the current study,

Table 4. Albumin concentration (g/L) before (prechallenge) and during immune system stimulation (ISS)1,2

Dietary M:M+C4

Period3

Prechallenge ISS0.42 38.33 ± 1.19a 33.67 ± 1.19a

0.47 31.96 ± 0.93b 28.76 ± 0.93b

0.52 32.59 ± 1.04b 27.84 ± 1.04b

0.57 34.32 ± 1.49b 29.32 ± 1.49b

0.62 32.67 ± 1.19b 29.33 ± 1.19b

a,bValues within columns followed by different superscripts differ (P < 0.05).1Immune system stimulation (ISS) was induced with increasing doses of

Escherichia coli lipopolysaccharide on d 1, 3, 5, and 7 of the ISS period. Values are greater during the prechallenge period than during the ISS (P < 0.05) and no interactive effect of ISS and dietary treatment was observed.

2The data presented are means ± SE (n = 15 to 17 pigs) and based on blood samples taken 1 d before ISS and d 8 after the start of ISS.

3Periods represent d 5 before ISS (prechallenge) and d 7 after start of ISS.4M:M+C = Met to Met plus Cys ratio.

Figure 1. Whole body protein deposition (Pd; g/day) in growing pigs fed varying dietary Met to Met plus Cys ratios (M:M+C) during the prechallenge period (A), immune system stimulation (ISS) period 1 (B), and ISS period 2 (C). Based on the quadratic-plateau analysis, a breakpoint was determined at 0.57 ± 0.03 M:M+C during the prechallenge period (A) and at 0.59 ± 0.02 M:M+C during ISS period 2 (C), representing the optimal M:M+C. No breakpoint was determined within the range of M:M+C diets evaluated in ISS period 1 (B).




in which daily M+C was maintained constant and only M:M+C was varied across treatments.

Second, ISS may also increase the need for Met (Yu et al., 1993). Methionine plays an important role as a methyl donor for processes such as DNA methylation and polyamine synthesis (Grimble, 2002; van de Poll et al., 2006). These roles become increasingly important during ISS to support the proliferation of immune cells (Dwyer, 1979). In addition, Met can be used as a catalytic antioxidant through the Met sulfoxide reductase system (Metayer et al., 2008), in which damaged proteins are repaired.

Lastly, Cys is extremely unstable and rapidly oxidizes to cystine (Meister, 1988). This process releases free radicals, contributing to the toxic effects of Cys (Meister, 1988; Grimble, 2002). In fact, the observed slight reduction in feed intake of pigs on the lowest M:M+C in the present study may be attributed to the toxic effect of Cys. The finding that the rate of TS in rats is increased during ISS further supports this hypothesis (Malmezat et al., 2000b). Although the rate of TS during ISS has not been directly measured in pigs, the activity of genes and enzymes involved in the TS pathway has been examined in pigs as well as humans and appears to favor TS during oxidative stress (Chen and Banerjee, 1998; Taoka et al., 1998; Malmezat et al., 2000a; Mosharov et al., 2000; Lu, 2009; Rakhshandeh

et al., 2010b,c). Therefore, the increase in optimum dietary M:M+C during ISS indicates that the dietary requirement for Met, relative to Cys, appears to be greater in pigs with disease.

It may be argued that part of the impact of ISS on the optimum dietary M:M+C can be attributed to changes in feed intake and growth performance and not to ISS per se. However, according to NRC (2012), a 20% reduction in feed intake, which is associated with a 29% reduction in BW gain, reduces the dietary M:M+C requirements by less than 1%. The latter is in contrast to the observed increase in the optimum dietary M:M+C observed in the current study.

In conclusion, the results of the present study indicate that the optimal dietary M:M+C is increased during ISS in growing pigs. This may be due to the inherent increased need for Met as a methyl donor and an antioxidant in the Met sulfoxide reductase system or due to the increased TS of Met to Cys for the synthesis of compounds involved in the immune response, such as GSH and APP. The current findings support the development of optimal dietary AA profiles for diseased pigs. However, further research should be conducted with dietary M:M+C that exceed 0.62 to determine the optimal ratio in ISS growing pigs because the response to increasing M:M+C was maintained at the greatest dietary M:M+C that was evaluated in the current study.

Table 5. Nitrogen balance and whole body protein deposition (Pd; N retention × 6.25) in growing pigs fed varying dietary methionine to methionine plus cysteine ratios (M:M+C) before (prechallenge) and during immune system stimulation (ISS)1

Item

Dietary M:M+C P-value0.42 0.47 0.52 0.57 0.62 Diet Lin2 Quad3

No. of pigsPrechallenge 6 7 5 6 8ISS period 1 5 7 6 5 8ISS period 2 6 7 6 6 8

N intake, g/dPrechallenge 14.2 ± 1.1 16.1 ± 0.1 16.2 ± 0.2 16.6 ± 0.1 16.9 ± 0.1 0.12 0.11 0.31ISS period 1 14.7 ± 0.2 15.9 ± 0.2 16.3 ± 0.2 16.5 ± 0.2 16.9 ± 0.2 0.001 <0.001 0.06ISS period 2 13.0 ± 1.4 15.6 ± 0.4 16.1 ± 0.4 16.5 ± 0.4 16.9 ± 0.4 0.21 0.09 0.25

Fecal N excretion, g/dPrechallenge 1.06 ± 0.13 1.21 ± 0.13 1.10 ± 0.14 1.20 ± 0.14 1.10 ± 0.12 0.86 0.85 0.52ISS period 1 1.33 ± 0.15 1.32 ± 0.12 1.45 ± 0.13 1.32 ± 0.15 1.13 ± 0.12 0.50 0.35 0.23ISS period 2 1.13 ± 0.11 1.11 ± 0.11 1.09 ± 0.11 1.20 ± 0.11 1.09 ± 0.10 0.93 0.96 0.89

Urinary N excretion, g/dPrechallenge 5.59 ± 0.25 5.06 ± 0.24 4.78 ± 0.26 4.62 ± 0.25 4.79 ± 0.24 0.002 0.001 0.02ISS period 1 6.42 ± 0.39 6.38 ± 0.28 6.23 ± 0.31 5.55 ± 0.34 5.93 ± 0.31 0.43 0.20 0.81ISS period 2 4.61 ± 0.72 5.20 ± 0.29 5.05 ± 0.35 4.77 ± 0.23 5.13 ± 0.20 0.70 0.73 0.75

Pd, g/dPrechallenge 50.5 ± 1.4 61.3 ± 1.3 64.8 ± 1.5 67.6 ± 1.4 68.3 ± 1.2 <0.001 <0.001 0.001ISS period 1 43.6 ± 1.9 51.2 ± 1.6 53.6 ± 1.7 60.4 ± 1.9 61.6 ± 1.5 <0.001 <0.001 0.20ISS period 2 47.9 ± 2.4 58.2 ± 2.2 62.2 ± 2.4 65.9 ± 2.4 66.6 ± 2.1 <0.001 <0.001 0.031Data presented are means ± SE and represent measurements taken in the 5 d period immediately before immune system stimulation (prechallenge) and during

immune system stimulation. Tolerance to lipopolysaccharide injection was observed at 4 d after initial injection; thus, N balance during ISS was evaluated over 2 periods (ISS period 1 and 3 d; ISS period 2 and 4 d).

2Lin = linear effect of dietary M:M+C.3Quad = quadratic effect of dietary M:M+C.




Moreover, the impact of different types of disease or ISS on Met and Cys requirements may be explored.

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