Effects of Supplementation of Mineral Nano Particles on Weaned Piglet Growth
presentación metabolismo
1. Immune system stimulation increases the optimal dietary methionine to methionine
plus cysteine ratio in growing pigs
N. Litvak, A. Rakhshandeh, J. K. Htoo and C. F. M. de Lange
J ANIM SCI 2013, 91:4188-4196.
doi: 10.2527/jas.2012-6160 originally published online July 3, 2013
The online version of this article, along with updated information and services, is located on
the World Wide Web at:
http://www.journalofanimalscience.org/content/91/9/4188
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3. 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
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+C
0.42 0.62
Cornstarch 48.64 48.71
Sucrose 20.00 20.00
Sodium caseinate 4.70 4.70
Cellulose 4.00 4.00
Corn oil 4.00 4.00
Limestone 0.80 0.80
Dicalcium phosphate 3.00 3.00
Salt 0.40 0.40
Potassium chloride 0.90 0.90
Magnesium sulfate 0.35 0.35
Vitamin–mineral mix2 0.50 0.50
Titanium dioxide 0.10 0.10
dl-Met 0.00 0.07
l-Cys HCl 0.23 0.15
l-Lys HCl 0.49 0.49
l-Thr 0.35 0.35
l-Trp 0.12 0.12
l-Ile 0.29 0.29
l-Val 0.29 0.29
l-His 0.15 0.15
l-Phe 0.46 0.46
l-Leu 0.46 0.46
l-Asp 4.83 4.83
l-Glu 4.88 4.88
Choline 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).
(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
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4. 4190 Authors Litvak et al.
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 Observations
Before 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
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5. Methionine and cysteine ratio for growing pigs 4191
Table 2. Calculated and analyzed nutrient contents (%, as-is basis) in the experimental diets1,2
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 <
Item
Dietary M:M+C
0.42 0.47 0.52 0.57 0.62
Anal Calc Anal Calc Anal Calc Anal Calc Anal Calc
CP 11.9 11.2 13.3 11.2 12.9 11.2 12.5 11.2 12.4 11.2
Met 0.12 0.13 0.15 0.14 0.17 0.16 0.18 0.18 0.18 0.19
Cys 0.16 0.17 0.15 0.16 0.14 0.15 0.13 0.13 0.12 0.12
Met + Cys 0.27 0.30 0.31 0.30 0.31 0.31 0.31 0.31 0.31 0.31
Lys 0.77 0.73 0.76 0.73 0.77 0.73 0.84 0.73 0.75 0.73
Thr 0.47 0.53 0.52 0.53 0.56 0.53 0.50 0.53 0.52 0.53
Trp 0.12 0.17 0.14 0.17 0.16 0.17 0.15 0.17 0.15 0.17
Ile 0.46 0.51 0.52 0.51 0.49 0.51 0.49 0.51 0.47 0.51
Leu 0.83 0.87 0.91 0.87 0.91 0.87 0.88 0.87 0.87 0.87
Val 0.55 0.58 0.59 0.58 0.56 0.58 0.57 0.58 0.55 0.58
His 0.25 0.28 0.28 0.28 0.29 0.28 0.27 0.28 0.27 0.28
Phe 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.
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6. 4192 Authors Litvak et al.
Table 3. Plasma concentrations of acute phase proteins and white blood cell count before (prechallenge) and during
immune system stimulation (ISS)1,2
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
Item
Period3 P-value
Prechallenge ISS Diet Period Diet × period
WBC4 count (× 109/L) 22.4 ± 1.6 22.0 ± 0.7 0.46 0.82 0.96
Haptoglobin, g/L 0.41 ± 0.07 0.90 ± 0.07 0.68 <0.001 0.70
Albumin, g/L 34.0 ± 0.5 29.8 ± 0.5 0.01 <0.001 0.67
Fibrinogen, 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.
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7. Methionine and cysteine ratio for growing pigs 4193
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 ISS
0.42 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).
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8. 4194 Authors Litvak et al.
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
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.
Item
Dietary M:M+C P-value
0.42 0.47 0.52 0.57 0.62 Diet Lin2 Quad3
No. of pigs
Prechallenge 6 7 5 6 8
ISS period 1 5 7 6 5 8
ISS period 2 6 7 6 6 8
N intake, g/d
Prechallenge 14.2 ± 1.1 16.1 ± 0.1 16.2 ± 0.2 16.6 ± 0.1 16.9 ± 0.1 0.12 0.11 0.31
ISS 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 0.06
ISS 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/d
Prechallenge 1.06 ± 0.13 1.21 ± 0.13 1.10 ± 0.14 1.20 ± 0.14 1.10 ± 0.12 0.86 0.85 0.52
ISS 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 0.23
ISS 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/d
Prechallenge 5.59 ± 0.25 5.06 ± 0.24 4.78 ± 0.26 4.62 ± 0.25 4.79 ± 0.24 0.002 0.001 0.02
ISS 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 0.81
ISS 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/d
Prechallenge 50.5 ± 1.4 61.3 ± 1.3 64.8 ± 1.5 67.6 ± 1.4 68.3 ± 1.2 <0.001 <0.001 0.001
ISS 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 0.20
ISS 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 0.03
1Data 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.
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9. Methionine and cysteine ratio for growing pigs 4195
Moreover, the impact of different types of disease or ISS
on Met and Cys requirements may be explored.
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11. References
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