Download Bioefficacy of L-lysine sulfate compared to feed grade lysine·HCl in... M. R. Smiricky , I. Mavromichalis , D. M. Albin

Bioefficacy of L-lysine sulfate compared to feed grade lysine·HCl in young pigs
M. R. Smirickya,*, I. Mavromichalisa,1, D. M. Albina, J. E. Wubbena, M. Rademacherb,
and V. M. Gaberta,2
a
Department of Animal Sciences, University of Illinois, 1207 West Gregory Drive, 180
Animal Sciences Laboratory, Urbana, IL 61801
b
Degussa-Hüls AG, D-63457 Hanau-Wolfgang, Germany
2
Abstract
A pig growth assay was conducted to determine the relative biological value
(RBV) of lysine from L-lysine sulfate relative to feed grade lysine·HCl. A maize-peanut
meal diet containing 6.2 g/kg total lysine was supplemented with 2 levels (1 and 2 g/kg)
of lysine from L-lysine·HCl or L-lysine sulfate. The RBV of L-lysine sulfate was
determined using multiple regression slope-ratio methodology, with gain and feed
efficiency as the response criteria. At the tested levels, linear responses for gain and feed
efficiency were obtained from increments of lysine from the two lysine sources. When
weight gain was regressed on supplemental lysine intake, the RBV of lysine in L-lysine
sulfate was 99% of the RBV of lysine in L-lysine·HCl. For feed efficiency was regressed
on supplemental lysine intake, the RBV of lysine in L-lysine sulfate was 97% of the RBV
of lysine in L-lysine·HCl. The t-test analysis revealed that the RBV of lysine in L-lysine
sulfate was not significantly different from the RBV of lysine in L-lysine·HCl, which was
assumed to be 100% bioavailable.
Key Words: Pigs, L-lysine·HCl, L-lysine Sulfate, Relative Bioavailability
Introduction
Lysine is accepted as the first limiting amino acid in pig diets based on maize and
soybean meal, and therefore it has become an established practice to supplement pig diets
with crystalline lysine in the form of L-lysine·HCl in order to meet the lysine requirement
of 13.5 g/kg (total basis) in rations for pigs weighing 5-10 kg (NRC, 1998). Recently, a
new source of L-lysine has been developed by Degussa-Hüls called Biolys 60. Biolys 60
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is a L-lysine sulfate product that contains byproducts from fermentation, with a minimum
lysine content of 473 g/kg. Although, L-lysine sulfate is a product of bacterial
fermentation of carbohydrates, like L-lysine·HCl, further processing methods differ
(Schutte and Pack, 1994). This new source of lysine is not anticipated to differ from the
standard L-lysine·HCl. However, due to the presence of by-products of fermentation or
otherwise known as dried microbial cells, differences in performance may be observed.
Whittemore and Moffat (1976) determined that DMC contained 18.8 MJ of DE/kg DM
and 119 g digestible N/kg DM for pigs.
Ammerman et al. (1995) defined “bioavailability” as the “degree to which an
ingested nutrient in a particular source is absorbed in a form that can be utilized in
metabolism by the animal”. Izquierdo et al. (1988) determined that crystalline Llysine·HCl is 100% bioavailable. A variety of methods are used to determine the
bioavailability of amino acids. However, measuring amino acid availability via growth
assays determines the ability of a protein to provide a specific limiting amino acid and
subsequently promote growth (Lewis and Bayley, 1995). A commonly used approach to
determine amino acid bioavailability is the slope-ratio technique (Batterham, 1992).
Therefore, objective of the current study was to compare the biological efficacy of Llysine sulfate with that of L-lysine·HCl in young pigs.
Materials and Methods
General. The University of Illinois Laboratory Animal Care and Use Committee
approved all experimental procedures (Protocol No. A8R197). Prior to the initiation of
the study, pigs were weaned at 21 d of age and were fed a 220 g/kg CP phase 1 nursery
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diet for approximately 7 d until they reached approximately 10 kg BW. One hundred
nursery pigs (Line 337 sire × C15 dams; PIC; Franklin, KY) with an average initial BW
of 9.5 ± 1.5 kg were randomly allotted to five dietary treatments in five replicates of four
pigs per pen based upon weight and gender. Pigs were removed from study after 3 wk on
test. Pigs were housed in an environmentally-controlled nursery facility with 100%
raised expanded metal flooring and fluorescent lighting. Each pen was equipped with a
five-hole feeder and one nipple waterer. Feed and water were provided for ad libitum
consumption. Pigs and feeders were weighed weekly to determine weight gain, feed
disappearance, and feed efficiency.
The basal diet (Table 1) met or exceeded requirements (NRC, 1998) for all nutrients
except amino acids. It was fortified with crystalline amino acids to reach 120% of the
ideal amino acid concentrations (Baker, 1997; NRC, 1998) with the exception of lysine.
Peanut meal was used as a protein source that was relatively balanced in all essential
amino acids, with the exception of lysine. The basal diet was then supplemented with 2
doses (1 and 2 g/kg) of lysine either as L-lysine·HCl or L-lysine sulfate at the expense of
maize starch. The L-lysine sulfate product, Biolys 60, contained at least 468 g/kg free Llysine and an additional 5 g/kg lysine bound in biomass, thus resulting in a total lysine
concentration of 473 g/kg. L-lysine·HCl contained 785 g/kg total lysine. The analyzed
free lysine content of the diets was used in all calculations.
Chemical analysis. Crude protein content in the experimental diets was determined by
the combustion technique using a Leco analyzer and the method of AOAC 990.03
(AOAC, 1995). Total amino acid content of the diets was quantified by ion-exchange
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chromatography with post-column derivation with ninhydrin following 24-hr acid
hydrolysis at 105°C with 6N HCl (Llames and Fontaine, 1994). Performic acid
oxidation preceded acid hydrolysis for the determination of methionine and cystine. All
other amino acids other than lysine were determined to verify that the diets contained
120% of the ideal amino acid concentrations. The determination of non-protein bound or
supplemental lysine in the experimental diets was quantified by ion-exchange
chromatography with post-column derivation with ninhydrin after hydrolysis with dilute
hydrochloric acid at room temperature using norleucine as an internal standard (Fontaine,
1995). Non-protein bound lysine analysis verified that the diets contained the correct
amount of added lysine for either L-lysine sulfate or L-lysine·HCl.
Statistical analysis. Pen means were analyzed as a randomized complete-block design
using the GLM procedure of SAS (1995), with dietary treatment and block as defined
sources of variation. Data was fitted in a multivariate linear regression model with the
equation y = a + b1x1 + b2x2, where a = common y-intercept of the 2 lines, b1 = slope of
L-lysine·HCl line, b2 = slope of L-lysine sulfate line, x1 = value for L-lysine·HCl, and x2
= value for L-lysine sulfate (Littell et al., 1995). The multiple regression model
consisted of 2 straight lines with a common intercept. The dependant variables, weight
gain and gain: feed, were regressed on supplemental lysine intake. The RBV was defined
as RBV = x2/x1 x 100, where x1 = L-lysine·HCl and x2 = L-lysine sulfate. An unpaired ttest was conducted to determine if the RBV of lysine in L-lysine sulfate was different
from the RBV of lysine in L-lysine·HCl (Petrie and Watson, 1999).
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Results
Supplementation of the basal diet with lysine increased (P < 0.05) in weight gain
and feed efficiency (Table 2). However, the source of lysine (L-lysine·HCl or L-lysine
sulfate) did not affect these parameters. Feed intake was also not affected by dietary
treatment. By supplementing the basal diet with 1 g/kg lysine, ADG increased 51% and
42% for the L-lysine·HCl and L-lysine sulfate diets, respectively. Feed efficiency was
improved 105% and 80 % for the L-lysine·HCl- and L-lysine sulfate-based diets,
respectively, compared to the basal diet. By supplementing the basal diet with 2 g/kg
lysine, ADG increased 165% and 202% for the L-lysine·HCl- and L-lysine sulfate-based
diets. Feed efficiency was improved 174% and 211% for the L-lysine·HCl- and L-lysine
sulfate-based diets, respectively, compared to the basal diet. An unpaired t-test was
conducted to determine if the slopes of the lines in Figures 1 and 2 were statistically
different from each other. The null hypothesis was that the slopes of the lines were
significantly different at P < 0.05. This test resulted in rejection of the null hypothesis
and proved the slopes of the L-lysine sulfate versus L-lysine·HCl lines were not different.
Relative biological value (RBV) was calculated for lysine in L-lysine sulfate. Slope
ratio analysis of weight gain showed that the RBV of lysine in L-lysine sulfate was 99%
(Figure 1). The analysis showed that the RBV of lysine in L-lysine sulfate was 97% of
that in L-lysine·HCl, based on feed efficiency (Figure 2). However, neither 99% nor 97%
were different (P > 0.20) from 100% as determined by an unpaired t-test (Petrie and
Watson, 1999).
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Discussion
In typical corn-soybean meal based diets for young pigs, lysine is the first limiting
amino acid (Mavromichalis et al., 1998). Therefore, lysine addition to these diets has
become a common practice in the swine industry. L-lysine·HCl is the dominant source of
lysine for addition to pig diet and is produced by bacterial fermentation of carbohydrates
and other ingredients (Nhan et al., 1976). After fermentation, cell separation occurs and
the biomass is removed. The chloride ion is then added to the lysine via ion exchange,
evaporation occurs and ammonia is released. After crystallization, the hydrochloric salt
product is dried to form L-lysine·HCl (Schutte and Pack, 1994), which contains about
785 g/kg free lysine.
L-lysine sulfate is produced via the same fermentation process. However, after
fermentation the biomass is not separated from the fermentation broth and the product is
maintained in the sulfate form. The resulting product then undergoes evaporation and
granulation. L-lysine sulfate contains 150 g/kg sulfate, and a small amount of other
nutrients, such as amino acids other than lysine, and phosphorus.
A preliminary comparison of L-lysine·HCl and L-lysine sulfate showed no
difference in their biological efficacy pigs (Schutte and Pack, 1994). Kirchgessner and
Roth (1996) also reported no significant differences in weight gain or feed efficiency in
pigs offered diets containing the same concentration of lysine as either L-lysine sulfate or
L-lysine·HCl.
Determination of bioavailability is important for accurate feed formulation and
maximal growth. L-lysine sulfate is a commercially available source of lysine that can be
used in swine feed formulation. Supplementation of the basal diet with lysine resulted in
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an increase in both ADG and feed efficiency (Table 2). However, the source of lysine
did not affect these response parameters. This is in agreement with an earlier study
conducted by Kirchgessner and Roth (1996) where supplementation of the deficient basal
diet with 1 or 2 g/kg lysine improved gain and feed efficiency, irrespective of lysine
source. Additionally, Fuller et al. (1986) reported that supplementation of their lysine
deficient diet significantly improved daily gain and feed efficiency regardless of the
source of lysine, either free L-lysine·HCl or soya bean meal.
The improvements in weight gain and feed efficiency can be attributed only to the
supplementation with lysine, because the experimental diets were formulated at 120% of
the ideal amino acid ratio (Baker, 1997). Furthermore, the experimental diets contained
2.7 g/kg Na and 4 g/kg Cl, which are above the NRC (1998) requirements. Mahan et al.
(1996) reported no improvement in weight gain as a result of dietary supplementation
with either Na or Cl after 14 d postweaning in starter pigs weaned at 23 ± 2 d of age
above the NRC recommended level. NRC (1998) recommends 1.5 g/kg of both Na and
Cl for pigs between 10 to 20 kg BW, and this study did not begin until 15 d postweaning.
These results suggest, therefore, that the experimental diets in the current study were
adequate in Na and Cl for growth in starter pigs, and any growth responses were the
result of lysine supplementation alone.
The RBV was determined by regressing gain on intake of supplemental lysine.
Baker (1986) suggested that bioavailability studies should be regressed on absolute intake
of the nutrient because otherwise variation in feed intake may affect bioavailability
results. Additionally, the nutrient intake should be in the constant slope region of the
growth curve or approximately 30 to 70% of animal requirement (Baker, 1986). The test
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diets provided 54 and 63% of the lysine requirement. Therefore, the supplemental lysine
levels are clearly deficient and fall in the linear portion of the growth curve. The RBV of
lysine in L-lysine sulfate did not differ from that of L-lysine·HCl. This lack of differnece
in RBV was additionally supported by the absence of differences in weight gain and feed
efficiency between the two lysine sources.
Conclusions
In conclusion, the bioavailability of lysine in L-lysine sulfate in promoting growth
in young pigs is no different from the lysine supplied by L-lysine·HCl. The RBV of
lysine in L- lysine·HCl was not different from the RBV of lysine in L-lysine sulfate from
Biolys 60. Therefore, in lysine deficient corn-SBM-based swine diets, Biolys 60 can be
used instead of L-lysine·HCl to fortify these diets.
References
Ammerman, C. B. 1995. Introduction. In: Ammerman, C. B., Baker, D. H., Lewis, A. J.,
(Eds.), Bioavailability of nutrients for animals: Amino Acids, Minerals, and Vitamins.
Academic Press, San Diego, CA, pp. 1-3.
AOAC. 1995. Official Methods of Analysis (16th Ed.). Association of Official Analytical
Chemists, Arlington, VA.
Baker, D. H. 1986. Problems and pitfalls in animal experiments designed to establish
nutrient requirement for essential nutrients. J. Nutr. 116: 2339-2349.
10
Baker, D. H. 1997. Ideal amino acid profiles for swine and poultry and their applications
in feed formulation. In: Biokyowa Technical Review-9, Nutri-Quest, Inc., Chesterfield,
MO, pp. 1-21.
Batterham, E. S. 1992. Availability and utilization of amino acids for growing pigs. Nutr.
Res. Rev. 5: 1-18.
D’Mello, J.P.F., Peers D. G., Whittemore, C. T. 1976. Utilization of dried microbial
cells grown on methanol in a semi-purified diet for growing pigs. Br. J. Nutr. 36: 403410.
Fontaine, J. 1995. Assays for amino acids: standardising methods throughout the EU.
Feed Int. 16: 16-21.
Fuller, M. F., Wood, J., Brewer, A. C., Pennie, K., MacWilliam R. 1986. The
responses of growing pigs to dietary lysine, as free lysine hydrochloride or in soya-bean
meal, and the influence of food intake. Anim. Prod. 43: 477-484.
Izquierdo, O. A., Parsons, C. M., Baker, D. H. 1988. Bioavailability of lysine in Llysine·HCl. J. Anim. Sci. 66: 2590-2597.
Kirchgessner, M., Roth, F. X. 1996. Comparsion of Biolys 60 vs. L-lysine·HCl in
11
piglet diets. In: Feedback facts and figures. Tech. Bull. No. 1. Degussa-Hüls, Hanau,
Germany.
Kirchgessner, M., Roth, F. X. 1996. Comparison of Biolys 60 vs. L-lysine·HCl in
piglet diets. In: Feedback facts and figures. Tech. Bull. No. 2. Degussa-Hüls, Hanau,
Germany.
Lewis, A. J., Bayley, H. S. 1995. Amino acid bioavailability. In: Ammerman, C. B.,
Baker, D. H., Lewis, A. J., (Eds.), Bioavailability of nutrients for animals: Amino Acids,
Minerals, and Vitamins. Academic Press, San Diego, CA, pp. 35-65.
Littell, R. C., Henry, P. R., Lewis, A. J., Ammerman, C. B. 1997. Estimation of
relative bioavailability of nutrients using SAS procedures. J. Anim. Sci. 75: 2672-2683.
Llames, C. R., Fontaine, J. 1994. Determination of amino acids in feeds:
collaborative study. J. Assoc. Off. Anal. Chem. 77: 1362-1402.
Mavromichalis, I., Webel, D. M., Emmert, J. L., Moser, R. L., Baker, D. H. 1998.
Limiting order of amino acids in a low-protein corn-soybean meal-whey-based diet for
nursery pigs. J. Anim. Sci. 76: 2833-2837.
Mahan, D. C., Newton, E. A., Cera, K. R. 1996. Effect of supplemental sodium
12
chloride, sodium phosphate, or hydrochloric acid in starter pig diets containing dried
whey. J. Anim. Sci. 74:1217-1222.
Nhan, H. B., Siehr, D. J., Findley M. E. 1976. Studies on the rate of lysine production
by Brevibacterium lactofermentum from glucose. J. Gen. Appl. Microbiol. 22: 65-78.
NRC, 1998. Nutrient Requirements of Swine. 10th ed. National Academy Press,
Washington, DC.
Petrie, A., Watson, P. 1999. Statistics for Veterinary and Animal Science. Blackwell
Science, Malden, MA.
SAS. 1995. SAS/STAT User’s Guide (Release 6.12). SAS Inst. Inc., Cary, NC.
Schutte, J. B., Pack, M. 1994. Biological efficacy of L-lysine preparations containing
biomass compared to L-lysine·HCl. Arch. Anim. Nutr. 46: 261-268.
Whittemore, C. T., Moffat, I. W. 1976. The digestibility of dried microbial cells
grown on methanol in diets for growing pigs. J. Agric. Sci., Camb. 86: 407-410.
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Table 1. Composition and nutrient analysis of the basal diet (fresh weight basis)
Ingredient
%
Corn
51.22
Peanut meal
37.38
Lactose
3.50
Animal/vegetable fat
3.00
Dicalcium phosphate
1.63
Limestone
1.04
Cornstarch
0.64
Vitamin premixa
0.20
Trace mineral premixb
0.35
Copper sulfate
0.08
DL-Methionine
0.17
L-Tryptophan
0.07
L-Threonine
0.22
Antimicrobial agentc
0.50
Calculated compositiond
ME, kcal/kg
3380
Ca, %
0.85
Available P, %
0.35
Na, %
0.15
Cl, %
0.22
Analyzed composition (%)e
CP
19.96
Lys
0.62
Thr
0.74
Met
0.38
14
Cys
0.29
Val
0.81
a
Supplied per kg of complete diet: retinyl acetate, 3410 µg; cholecalciferol, 25 µg; dl-
α-tocopheryl acetate, 132 mg; menadione sodium bisulfite complex, 7 mg; niacin, 50 mg;
d-Ca-pantothenate, 36 mg; riboflavin, 13 mg; vitamin B12, 53 µg; choline chloride, 486
mg; folate, 4mg.
b
Supplied per kg complete diet: Fe, 90 mg (FeSO4•H2O); Zn, 100 mg (ZnO); Mn, 20
mg (MnO); Cu, 8 mg (CuSO4); I, 0.35 mg (CaI2); Se, 0.3 mg (Na2SeO3); NaCl, 3 g.
c
Provided per kg complete diet: 110 mg chlortetracycline, 110 mg of sulfamethazine,
and 55 mg of penicillin.
d
e
Calculated (NRC, 1998).
Analyzed (Llames and Fontaine, 1994)
15
Table 2. Response parameters of pigs fed different lysine sources
Treatments
1. Basal diet (B)
2. B + 0.1% lysine
from L-lysine·HCl
3. B + 0.2% lysine
from L-lysine·HCl
4. B + 0.1% lysine
from L-lysine sulfate
5. B + 0.2% lysine
from L-lysine sulfate
Pooled SEM
a
Supplemental
lysine intake,
mg/db
Weight
gain,
g/dc
Feed
intake,
g/dc
Feed
efficiency,
g/gc
0
85
829
0.104
1.0
815
173
815
0.214
2.2
1743
225
792
0.285
1.2
956
147
797
0.187
2.3
1872
257
12
814
56
0.324
0.019
Determined by Degussa-Hüls AG, Germany using the method of Fontaine (1995).
b
c
Analyzed
supplemental
lys,
g/kga
0
Analyzed supplemental lysine in diet × feed intake.
Values are means for five pens of four pigs per pen. The initial BW of pigs was 9.45 ±
1.5 kg. The pigs were on test for 21 d.
Weight gain, g/d
16
300
200
100
Weight gain (g/d) = 88 + 0.083 × X1 + 0.082 × X2
0
0
1000
2000
Supplemental lysine intake, mg/d
C o n tro l
L -lys in e ·H C l
(X1)
L -lys in e s u lfa te
(X2)
Figure 1. Regression of weight gain against supplemental lysine intake from either Llysine·HCl or L-lysine sulfate. A total of 100 pigs (10 kg BW) were used in 4 replicates
in a 21-d growth assay. Relative bioavailability of lysine in L-lysine sulfate was 99%
compared to lysine in feed-grade L-lysine·HCl.
17
Gain: feed, g/kg
300
200
100
Gain: feed (g/kg) = 124 + 0.093 x X1 + 0.091 x X2
0
0
1000
Supplemental lysine intake, mg/d
C o n tro l
L -lys in e ·H C l
(X1)
2000
L -lys in e s u lfa te
(X2)
Figure 2. Regression of feed efficiency against supplemental lysine intake from either Llysine·HCl or L-lysine sulfate. A total of 100 pigs (10 kg BW) were used in 4 replicates
in a 21-d growth assay. Relative bioavailability of lysine in L-lysine sulfate was 97%
compared to lysine in feed-grade L-lysine·HCl.