Received: 24 February 2017 | Accepted: 18 August 2017

DOI: 10.1111/jpn.12813

ORIGINAL ARTICLE

2-­Hydroxy-­4-­methylthio butanoic acid and DL-­methionine for

Japanese quails in production

D. V. G. Vieira1 | F. G. P. Costa2 | M. R. Lima3 | J. G. de V. Júnior4 |

T. P. Bonaparte4 | D. T. Cavalcante2

1
Federal University of Tocantins, Araguaina,
Brazil Summary
2
Federal University of Paraiba, Areia, Brazil An experiment was performed using 1,000 laying Japanese quails to assess the avail-
3
Federal University of South Bahia, Teixeira de ability of two alternative dietary methionine sources. Treatment 01 = Basal Feed that
Freitas, Brazil
is deficient in digestible methionine + cystine (Met + Cys). The other treatments were
4
Federal University of Espirito Santo, Alegre,
Brazil constituted by Met + Cys levels of 0.8, 1.60 and 2.40 g/kg, supplemented with DL-­
Methionine-­99%, HMTBA-­88% and HMTBA-­84%, being 10 treatments in total. The
Correspondence
D. V. G. Vieira, Federal University of Tocantins, following characteristics were studied: feed intake (g/bird/day), egg production (egg/
University Campus Araguaina, School of day × 100), egg weight (g/egg), egg mass (g/egg), feed conversion per egg dozen (kg
Veterinary Medicine and Animal Science,
Araguaina/Tocantins, Brazil. feed/dozen eggs), feed conversion per egg mass (kg feed/kg eggs), relative yolk weight
Email: danilovargaszoo@homail.com (g/100 g of egg), relative albumen weight (g/100 g of egg), relative shell weight
(g/100 g of egg), shell thickness (mm) and specific gravity (g/cm3). In general result
comment, supplemental methionine sources must be included in the poultry diet. The
different methionine sources affect the performance of quails, and the increase in the
levels within each source improves the performance variables. Significant effect was
observable on performance variables and egg quality variables, being that DLM-­99%
is superior to the other sources. The HMTBA-­88% source is superior to the
HMTBA-­84% source for the same aforementioned variables. In conclusion, the bioef-
ficacy values of the HMTBA-­88% and HMTBA-­84% sources compared to the DLM-­
99% source on an equimolar basis were 81 and 79%, respectively, for the performance
variables, and 83 and 74 while the methionine sources were equivalent for the varia-
bles related to egg quality.

KEYWORDS
availability, bioefficacy, egg quality, hydroxy analogue, performance

1 | INTRODUCTION (Boomgardt & Baker, 1973). Dietary methionine levels may also change
egg weight and egg production (Costa et al., 2009; Reis et al., 2011).
Methionine stands out among the essential nutrients that directly af- New methionine sources are available on the market in liquid and
fect poultry performance as the first limiting amino acid for poultry fed powder forms. The most used source in diets, DL-­methionine (DLM-­
maize and soybean meal-­based diets. Methionine plays several roles 99%), is sold in powder form. The methionine hydroxy analogue
in the poultry organism and affects the immune system (Kalinowski, 2-­hydroxy-­4-­methylthio butanoic acid (HMTBA) is available in solution
Moran, & Wyatt, 2003), protein deposition (Hruby, 1998), lipid me- (88% methionine) and powder (84% methionine, calcium salt) forms.
tabolism (Jensen, Wyatt, & Fancher, 1989) and energy metabolism The alternative source HMTBA is an alpha-­keto acid of methionine;

J Anim Physiol Anim Nutr. 2017;1–9. wileyonlinelibrary.com/journal/jpn

© 2017 Blackwell Verlag GmbH | 1
2 | VIEIRA et al.

alpha-­keto acids are efficiently converted into their respective amino After reaching 40 days of age, the quails were housed in galvanised
acids by transamination (Nelson & Cox, 2014). wire cages (100 × 30 × 20 cm) stacked into three levels, arranged in a
The efficacy clearly depends upon the type of diet and supple- ladder pattern. Each cage had three partitions (33.33 × 30 × 20 cm)
mentation level, with supplemental, amino acid-­based, purified exper- wherein 10 quails were housed, corresponding to a density of 99 cm2/
imental diets apparently favouring DLM-­99%. However, poultry fed quail. Feeders (trough in front of the cage) and drinkers (nipple in the
commercial diets containing HMTBA may have better responses than rear of the cage) were used.
poultry fed diets with DLM-­99% at practical levels of supplementation After recording 5% production, the quails were fed diets that met
(Gonzales-­Esquerra et al., 2007; Vazquez-Anon, Kratzer, Gonzalez- the demands of the laying phase (Silva, Jordão Filho, Costa, Lacerda, &
Esquerra, Yi, & Knight, 2006; Vazquez-Anon, Gonzalez-Esquerra et al., Vargas, 2012) and received light stimulation, with one light hour added
2006). This efficacy is such that HMTBA is used as a methionine per week until 16 hr of light. The egg-­laying evaluation period began
source in the poultry industry in United States of America, with an 85 in the week when 16 hr of light exposure was reached to standardise
to 88% bioefficacy level for liquid methionine. the lot according to egg production during a 10-­day period. After this
Much of the confusion generated around bioefficacy among nu- period, the quails were individually weighed and distributed according
tritionists results from deficient experimental protocols. Most experi- to weight and, subsequently, egg laying (Sakomura & Rostagno, 2007).
ments consider bioefficacy to be arbitrary, assigning a pre-­set efficacy The quails were weighed in the late afternoon to ensure they had all
value to the commercial products used in the comparison, that is, they performed egg laying on that day.
consider that the bioefficacy value of HMTBA-­88% is 65% and the A total of 10 treatments with 10 replicates with 10 quails per ex-
value of DLM-­99% is 100% and therefore proceed to the comparison perimental unit (1000 animals in total) were used. The treatments were
between the two products by formulating with this level of equiva- allocated to the respective experimental units according to an entirely
lence. In reality, diets should be formulated based on the concentra- randomised design. The bioefficacy (Table 1) of the methionine sources
tion of active products (equimolar comparison) to conduct a proper was compared based on the methionine supply of each source, that is,
comparison, that is, HMTBA-­88% and HMTBA-­84% and DLM-­99%. on an equimolar basis. Treatment 01 = Basal feed that was deficient
This way, experimental diets will have the same digestible methionine in digestible methionine + cystine. Treatments 02, 05 and 08 = Basal
concentration (Gonzales-­Esquerra et al., 2007; Vazquez-Anon, Kratzer diet + 0.80 g/kg digestible Met + Cys (Methionine + cystine) supplied
et al., 2006; Vazquez-Anon, Gonzalez-Esquerra et al., 2006). by the following sources: DLM-­99%, HMTBA-­88% and HMTBA-­84%
Several studies on bioefficacy of dietary methionine and its respectively. Treatments 03, 06 and 09 = Basal diet + 1.60 g/kg di-
sources in the diets of the birds, there is still limited information in gestible Met + Cys supplied by the following sources: DLM-­99%,
quails to DLM and HMTBA, and how this amino acid influences the HMTBA-­88% and HMTBA-­84% respectively. Treatments 04, 07
quail performance and egg quality. Thus, the aim was to assess this and 10 = Basal diet + 2.40 g/kg digestible Met + Cys supplied by
effect on Japanese quails during the egg production period. the following sources: DLM-­99%, HMTBA-­88% and HMTBA-­84%
respectively.
The amounts of the methionine sources in the diets (Table 1)
2 | MATERIALS AND METHODS
corresponded to supplementation with 0.8, 1.60 and 2.40 g/kg
more digestible Met + Cys than the Basal diet (5.624 g/kg), pro-
2.1 | Location and duration of the experiment
viding levels of digestible Met + Cys of 6.424 g/kg (T2, T5 and T8);
The experiment was conducted by the Research Group on Poultry 7.224 g/kg (T3, T6 and T9); and 8.024 g/kg (T4, T7 and T10). The
Technologies (Grupo de Estudos em Tecnologias Avícolas, GETA) at dietary values of metabolisable energy and crude protein were ad-
the facilities of the Campus II Poultry Farming Section of the Federal justed according to the amount of supplemental dietary digestible
University of Paraíba (Universidade Federal da Paraíba), located in methionine sources through the addition of l-­glutamic acid (crude
Areia, Paraíba (PB), Brazil. The experiment lasted 120 days, divided protein and metabolisable energy) and maize starch (metabolisable
into five periods of 24 days for data collection. The project had ethi- energy).
cal approval from the Animal Use and Care Committee of the Federal The diets samples were analysed for DM by placing triplicate
University of Paraiba, Brazil. samples in a drying oven at 105°C for 24 hr (AOAC, 1990). Nitrogen
content (triplicate samples) of feed samples were determined on a
0.25 g sample with a combustion analyser (Leco model FP-­2000N
2.2 | Installations and equipment
analyser) using EDTA as a calibration standard, with CP being cal-
A total of 1,500 one-­day-­old female Japanese quails (egg produc- culated by multiplying percentage N by 6.25. Complete AA content
tion) were purchased from the Fujikura® Company (Suzano, Sao of the diets was analysed by a commercial laboratory in triplicates
Paulo, Brazil). The quails were reared until the age of 40 days (on the for diets (Aoac, 1990; method 982.30 E (a, b, c). Performic acid ox-
ground). During that period, they received water ad libitum and two idation (Aoac, 1990; method 985.28) was conducted before acid
diets (1–21 and 22–42 days of age) formulated to meet the demands hydrolysis for the determination of Met and Cys, whereas all other
recommended in the Table for Japanese and European quails (Silva & AAs were determined after acid hydrolysis. Gross energy was de-
Costa, 2009). termined using an adiabatic bomb calorimeter (model 356, Parr
VIEIRA et al. | 3

TABLE 1 Experimental diets for Japanese quails

Basal DLM-­99% HMTBA-­88% HMTBA-­84%

Ingredients, g/100 g T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Corn 48.65 48.65 48.65 48.65 48.65 48.65 48.65 48.65 48.65 48.65
Soybean meal 37.26 37.26 37.26 37.26 37.26 37.26 37.26 37.26 37.26 37.26
Limestone 7.42 7.42 7.42 7.42 7.42 7.42 7.42 7.39 7.34 7.33
Soy oil 3.91 3.91 3.91 3.91 3.91 3.91 3.91 3.91 3.91 3.91
Dicalcium 1.02 1.02 1.02 1.02 1.02 1.02 1.02 1.02 1.02 1.02
phosphate
Salt 0.59 0.59 0.59 0.59 0.59 0.59 0.59 0.59 0.59 0.59
l-­valine 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
l-­threonine 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
Maize starch 0.21 0.16 0.12 0.08 0.17 0.14 0.11 0.17 0.13 0.09
l-­glutamic acid 0.39 0.30 0.22 0.13 0.30 0.20 0.11 0.30 0.22 0.14
DLM-­99% — 0.08 0.16 0.25 — — — — — —
HMTBA-­88% — — — — 0.09 0.18 0.27 — — —
HMTBA-­84% — — — — — — — 0.10 0.19 0.29
Choline chloride 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07
Vitamin premixa 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10
b
Minerals premix 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
Antioxidantc 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
d
Inert 0.27 0.32 0.36 0.41 0.31 0.34 0.37 0.33 0.41 0.44
Total 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
Analysis results nutritional composition
Crude protein, g/ 220.0 220.0 220.0 220.0 220.0 220.0 220.0 220.0 220.0 220.0
kg
MEe, kcal/kg 2,800 2,800 2,800 2,800 2,800 2,800 2,800 2,800 2,800 2,800
Calcium, g/kg 31.5 31.5 31.5 31.5 31.5 31.5 31.5 31.5 31.5 31.5
Available 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0
phosphorus, g/
kg
Sodium, g/kg 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
SID
Methionine + 5.6 6.4 7.2 8.0 6.4 7.2 8.0 6.4 7.2 8.0
cystine, g/kg
SID
Lysine, g/kg 10.5 10.5 10.5 10.5 10.5 10.5 10.5 10.5 10.5 10.5
SID
Threonine, g/kg 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3
SID
Valine, g/kg 9.4 9.4 9.4 9.4 9.4 9.4 9.4 9.4 9.4 9.4

SID, standardised ileal digestibility.

a
Composition/kg diet: Vit. A—12,000 IU; Vit. D3—3,600 IU; Vit. B1—2.5 mg; Vit. B2—8 mg; Vit. B6—3.0 mg; Pantothenic acid—12 mg; Biotin—0.2 mg; Vit.
K—3.0 mg; Folic acid—3.5 mg; Nicotinic acid—40 mg; Vit. B12—20mcg.
b
Composition/kg product: Mn—80 mg; Fe—50 mg; Zn—50 mg; Cu—10 mg; Co—1 mg; I—1 mg.
c
Etoxiquim.
d
Washed sand.
e
Metabolisable energy.

Instrument Company, Moline, IL, USA) and EE, after 3N HCl acid
2.3 | Variables evaluated
hydrolysis (method Am 5–04), using an Ankom XT10 extraction sys-
tem (Ankom Technology Corp. Macedon, NY, USA) as described by The following characteristics were studied: feed intake (g/quail/
AOCS (2004). In addition, the diets were analysed for phosphorus day), egg production (egg/day ×100), egg weight (g/egg), egg mass
and calcium (method 985.01) as described by AOAC International (g/egg), feed conversion per egg dozen (kg feed/dozen eggs), feed
(2000). conversion per egg mass (kg feed/kg eggs), relative yolk weight
4 | VIEIRA et al.

T A B L E 2 Feed intake (FI, g/quail/day), egg production (EP, %), egg weight (EW, g/egg), egg mass (EM, g/egg), feed conversion per egg mass
(FCEM, kg feed/kg egg) and feed conversion per egg dozen (FCED, kg feed/dozen) of Japanese quails fed diets containing different sources,
with orthogonal contrasts. Levels of digestible methionine + cystine for each source of methionine source in diet. And angular coefficient (β1)
and coefficient of determination (r2) of regression equations for the FI, EP, EW, EM, FCEM, FCED and bioavailability of sources HMTBA-­88%
and HMTBA-­84% relative to the default search DLM-­99%

Sourcea FI EP EW EM FCEM FCED

Basal diet—no source of methionine 30.56 79.90 10.39 8.30 3.68 0.459
Basal diet + DLM-­99% 28.09 92.11 11.57 10.68 2.66 0.367
Basal diet + HMTBA-­88% 29.22 88.86 10.91 9.72 3.05 0.396
Basal diet + HMTBA-­84% 29.40 88.00 10.83 9.56 3.12 0.403
p value 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001
SEM 1.527 5.411 0.703 1.167 0.503 0.044
Basal diet vs DLM-­99% 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001
Basal diet vs HMTBA-­88% 0.0001 0.0001 0.0020 0.0001 0.0001 0.0001
Basal diet vs HMTBA-­84% 0.0001 0.0001 0.0071 0.0001 0.0001 0.0001
DLM-­99% vs HMTBA-­88% 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001
DLM-­99% vs HMTBA-­84% 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001
HMTBA-­88% vs HMTBA-­84% 0.2456 0.0002 0.5047 0.1160 0.0309 0.0002
Basal diet + DLM-­99% + 0.08% 29.63 87.71 10.85 9.51 3.12 0.405
Met+Cys
Basal diet + DLM-­99% + 0.16% 28.11 91.84 11.82 10.85 2.59 0.367
Met+Cys
Basal diet + DLM-­99% + 0.24% 26.55 96.79 12.04 11.66 2.28 0.329
Met+Cys
p value b 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001
SEM 1.6107 6.3678 0.7189 1.3276 0.5517 0.0495
Basal diet + HMTBA-­88% + 0.08% 30.50 82.84 10.23 8.47 3.60 0.442
Met+Cys
Basal diet + HMTBA-­88% + 0.16% 29.55 90.42 10.82 9.79 3.02 0.392
Met+Cys
Basal diet + HMTBA-­88% + 0.24% 27.62 93.32 11.69 10.91 2.53 0.356
Met+Cys
p valuec 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001
SEM 1.2909 5.6112 0.6188 1.1051 0.4837 0.0424
Basal diet + HMTBA-­84% + 0.08% 30.98 81.56 10.38 8.46 3.66 0.456
Met+Cys
Basal diet + HMTBA-­84% + 0.16% 29.89 89.23 10.59 9.45 3.17 0.402
Met+Cys
Basal diet + HMTBA-­84% + 0.24% 27.34 93.21 11.54 10.76 2.54 0.352
Met+Cys
p valued 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001
SEM 1.4928 5.6443 0.6112 1.0569 0.4873 0.0454
CVe 2.17 2.25 2.77 2.89 2.96 2.45

DLM-­99% HMTBA-­88% HMTBA-­84% Bioavailability, %

2 2 2
Variables r β1 r β1 r β1 HMTBA-­88% HMTBA-­84%

FI 98.74 −16.917 84.37 −12.197 72.33 −13.422 72 79

EP 98.01 68.532 96.34 59.822 94.92 59.527 87 87
EW 94.91 7.412 78.53 5.627 73.60 4.60 76 62
EM 99.03 14.267 93.02 11.445 91.10 10.47 80 73
FCEM 98.48 −5.9187 93.23 −5.0317 88.66 −4.888 85 83

(Continues)
VIEIRA et al. | 5

TABLE 2 (Continued)

DLM-­99% HMTBA-­88% HMTBA-­84% Bioavailability, %

Variables r2 β1 r2 β1 r2 β1 HMTBA-­88% HMTBA-­84%

FCED 99.21 −0.5345 97.04 −0.450 90.92 −0.468 84 88

Average 81% 79%

SEM, Standard error mean; r2, coefficient of determination; β1, slope.

a
The total number of treatments was 10: Basal diet, three methionine supplementation levels of cystine (Met+Cys) for each source, thus satisfying the
prerequisite T—1 possible contrast. DLM-­99%—DL-­Methionine; HMTBA-­88%—Hydroxy analogue of methionine liquid; HMTBA-­84%—Hydroxy analogue
of methionine powder.
b
Linear effect for levels of DLM-­99%.
c
Linear effect levels HMTBA-­88%.
d
Linear effect levels HMTBA-­84%.
e
General coefficient of variation.

(g/100 g of egg), relative albumen weight (g/100 g of egg), relative the means of the DLM-­99%, HMTBA-­88% and HMTBA-­84% treat-
shell weight (g/100 g of egg), shell thickness (mm) and specific grav- ment groups respectively.
ity (g/cm3). Another analysis, polynomial regression, was performed consider-
To assess egg weight and its components, specific gravity and shell ing only the control diet and supplementation levels from the same
thickness (digital micrometre—Mitutoyo, 0.001 mm resolution), all eggs methionine source to assess the occurrence of a linear effect on the
from the last three days of each period (days 22, 23 and 24; 46, 47 and supplementation levels within each source (DLM-­99%; HMTBA-­88%
48; 70, 71 and 72; 94, 95 and 96; and 118, 119 and 120) were identi- and HMTBA-­84%).
fied for subsequent analyses, performed on the day of each collection. The ratios of the regression coefficients (Sasse & Baker, 1973)
The egg weight (eggs produced per experimental unit) was mea- were used to assess the bioefficacy of HMTBA-­88% and HMTBA-­84%
sured using a digital scale with an accuracy of 0.001 g. The production compared to DLM-­99%, adopting the following linear regression
was recorded daily in the morning. The specific gravity was assessed model: Y = α + βiXi + eijk, wherein: Y = observed values of the evaluated
immediately after weighing the eggs by immersion in buckets with dif- characteristics; α = intercept; βi = regression coefficients for each me-
ferent salt solutions (NaCl) at densities ranging from 1.060 to 1.095 g/ thionine source; and Xi = DLM-­99%, HMTBA-­88% and HMTBA-­84%
cm3 in 0.005 intervals, calibrated using a Petroleum densimeter. The supplemental levels.
eggs were then broken, and the yolk was separated and weighed. The
shells were washed and dried in a forced air oven for the subsequent
measurement of shell weight and thickness, which was performed in 3 | RESULTS AND DISCUSSION
the central region of the eggs. The albumen weight was calculated by
subtracting the shell and yolk weight from the egg weight. The methionine sources used in the diets (DLM-­99%; HMTBA-­88%
The quails that died during the experimental period were tallied and HMTBA-­84%) affected the performance of the quails (Table 2).
for subsequent adjustment of the feed intake and feed conversions, However, there was no significant effect comparing the two alterna-
according to the method reported by Sakomura and Rostagno (2007). tive sources, for the analysis of orthogonal contrasts (HMTBA-­88 vs
HMTBA-­84%) for the following variables: feed intake (p = .2456), egg
weight (p = .5047) and egg mass (p = .1160). The other variables egg
2.4 | Statistical analysis
production (p = .002) and feed conversion per egg dozen (p = .002)
The statistical analysis of the characteristics studied was performed were affected by the methionine sources present in the diets.
using the Statistical Analysis Software (SAS Institute, 1999). The anal- The performance of quails not fed a diet with a supplemental
ysis of variance (ANOVA) assumptions (error normality, random and methionine source (T01, Basal diet) was inferior to that of supple-
independent errors and variance homoscedasticity) were met. After mented quail, that is, they showed higher feed intake (30.56 g/quail/
this finding, the analysis was performed considering the 10 treat- day), lower egg production (79.90 egg/day × 100), lower egg weight
ments to evaluate the effect of using the different methionine sources (10.39 g/egg) and higher feed conversion per egg mass (3.68 kg feed/
with the following model: Y = μ + Ti + eijk, wherein μ = overall mean; kg eggs) and per egg dozen (0.459 kg feed/dozen eggs). This result
Ti = treatment effect; and eijk = random error associated with each explains the need for poultry supplementation with methionine, re-
observation in each treatment (i), period (j) and block (k). The treat- gardless of the source, because this amino acid is considered essential
ments were compared using orthogonal comparisons: Y1 = mi − (mj)/3; and is the first limiting amino acid for poultry, for both egg and meat
Y2 = mi − (mk)/3; Y3 = mi − (mf)/3; Y4 = (mj)/3 − (mk)/3; Y5 = (mj)/3 − production (Costa et al., 2009; Reis et al., 2011).
(mf)/3; Y6 = (mk)/3 − (mf)/3, wherein (i = 1) represents the mean of the Increased feed intake is observed when the quails are fed diets de-
control treatment; and j, k and f, with values of 1, 2 and 3, represent ficient in some nutrients (the amino acid methionine in this experiment)
6 | VIEIRA et al.

T A B L E 3 Shell thickness (ST, mm), relative yolk weight (YW, g/100 g of egg), relative shell weight (SW, g/100 g of egg), relative albumen
weight (AW, g/100 g of egg) and specific gravity (SG, g/cm3) of Japanese quail fed diets containing different sources of digestible methionine,
with orthogonal contrasts. Levels of digestible methionine + cystine for each source of methionine source in diet. And angular coefficient (β1)
and coefficient of determination (r2) of regression equations for the ST, YW, SW, AW and bioavailability of sources HMTBA-­88% and
HMTBA-­84% relative to the default font DLM-­99%

Sourcea ST YW SW AW SG

Basal diet—no source of methionine 205.04 36.00 9.53 54.47 1.070

Basal diet + DLM-­99% 205.84 32.28 8.85 58.79 1.072
Basal diet + HMTBA-­88% 205.31 34.30 9.48 56.22 1.073
Basal diet + HMTBA-­84% 204.75 34.30 9.51 56.18 1.072
p value 0.8990 0.0001 0.0001 0.0001 0.0142
SEM 2.2496 0.173 0.044 0.204 0.0013
Basal diet vs DLM-­99% 0.5166 0.0001 0.0001 0.0001 0.0014
Basal diet vs HMTBA-­88% 0.8255 0.0106 0.0246 0.7667 0.0003
Basal diet vs HMTBA-­84% 0.8108 0.0106 0.0273 0.9245 0.0048
DLM-­99% vs HMTBA-­88% 0.5443 0.0001 0.0001 0.0001 0.4384
DLM-­99% vs HMTBA-­84% 0.2128 0.0001 0.0001 0.0001 0.5347
HMTBA-­88% vs HMTBA-­84% 0.5164 0.9988 0.9499 0.7753 0.1666
Basal diet + DLM-­99% + 0.08% Met+Cys 205.89 34.34 9.40 56.26 1.0718
Basal diet + DLM-­99% + 0.16% Met+Cys 205.42 31.34 8.67 59.99 1.0724
Basal diet + DLM-­99% + 0.24% Met+Cys 206.21 31.15 8.49 60.36 1.0726
b
p value 0.1932 0.0001 0.0001 0.0001 0.001
SEM 0.9159 0.837 0.180 0.849 0.0011
Basal diet + HMTBA-­88% + 0.08% Met+Cys 205.46 36.71 10.05 53.24 7.0730
Basal diet + HMTBA-­88% + 0.16% Met+Cys 204.74 34.45 9.56 55.99 1.0716
Basal diet + HMTBA-­88% + 0.24% Met+Cys 205.73 31.75 8.83 59.42 1.0732
p valuec 0.3627 0.0001 0.0001 0.0001 0.0510
SEM 0.9326 0.986 0.254 1.110 0.0016
Basal diet + HMTBA-­84% + 0.08% Met+Cys 205.85 36.45 9.99 53.56 1.0716
Basal diet + HMTBA-­84% + 0.16% Met+Cys 204.11 34.18 9.58 56.24 1.0724
Basal diet + HMTBA-­84% + 0.24% Met+Cys 204.27 32.28 8.97 58.75 1.0720
d
p value 0.8584 0.004 0.009 0.004 0.0955
SEM 3.3398 1.681 0.406 1.985 0.0014
e
CV 2.15 3.61 3.34 2.54 0.11

DLM-­99% HMTBA-­88% HMTBA-­84% Bioavailability

Variables r2 β1 r2 β1 r2 β1 HMTBA-­88% HMTBA84%

ST 58.20 3.802 15.70 1.690 42.68 −5.057 — —

YW 91.69 −1.7532 77.71 −1.5005 82.83 −1.3436 85 77
SW 91.91 −0.3838 44.12 −0.2568 41.28 −0.2078 — —
AW 92.35 2.1390 72.14 1.7573 76.75 1.5514 82 72
SG 85.69 0.009 49.45 0.009 69.89 0.007 — —
Average 83% 74%

SEM, standard error mean; r2, coefficient of determination; β1, slope.

a
The total number of treatments was 10: Basal diet, three methionine supplementation levels of cystine (Met+Cys) for each source, thus satisfying the
prerequisite T—1 possible contrast. DLM-­99%—DL-­Methionine; HMTBA-­88%—Hydroxy analogue of methionine liquid; HMTBA-­84%—Hydroxy analogue
of methionine powder.
b
Linear effect for levels of DLM-­99%.
c
Linear effect levels HMTBA-­88%.
d
Linear effect levels HMTBA-­84%.
e
General coefficient of variation.
VIEIRA et al. | 7

(Basal diet) to meet daily nutrient requirements (Rodrigueiro, Albino, However, all methionine sources have both isomeric forms of the mol-
& Rostagno, 2000). This effect was observed through the following ecule (L and D), requiring the conversion of D into L forms for use in
comparisons: Basal diet vs. DLM-­99%; Basal diet vs. HMTBA-­88% and protein formation (Butolo, 2002). This last factor would not explain
Basal diet vs. HMTBA-­84% (Table 2). the difference in performance because all sources and their constit-
Some studies (Costa et al., 2009; Scottá et al., 2011) observed no uents would require conversion into the l-­methionine form. Different
differences in feed intake when studying different levels of dietary me- enzymes catalyse the oxidation of both isomers of the DLM-­99%,
thionine, albeit from a single source (DLM-­99%), even for diets with HMTBA-­88% and HMTBA-­84% sources (Knight & Dibner, 1984).
lower methionine levels 6.0 g/kg Met + Cys in the study by Scottá The difference in efficacy of the methionine sources could be re-
et al. (2011) and 5.5 g/kg Met + Cys in the study by Costa et al. (2009) lated to their intestinal absorption. DLM-­99% is actively absorbed,
in contrast with the result from this study, as the basal diet contained whereas the HMTBA sources are passively absorbed, and a small part
5.624 g/kg Met + Cys. may be performed by carriers (Suida, 2006). In addition to the differ-
The comparison of methionine sources shows that the source af- ence in absorptive processes, the intestinal macrobiotic seems to af-
fects the poultry’s feed intake, that is, animals fed supplemental di- fect the absorption and efficacy of methionine sources because there
etary methionine from the DLM-­99% source had a lower feed intake, is increased degradation of analogue sources by micro-­organisms
when comparing to HMTBA-­88% and HMTBA-­84%, indicating higher (Drew, Van Kessel, & Maenz, 2003). Furthermore, because HMTBA
efficacy of the DLM-­99% source, which will be discussed below. absorption is almost completely passive, it remains in the intestinal
Other studies, including Buresh and Harms (1986), Schutte and lumen longer, thus increasing the action of micro-­organisms. This
Weerden (1987) and Costa and Bastiani (1997), also found no sig- would not occur with DLM-­99% because this source is almost en-
nificant differences in feed intake when comparing two methionine tirely absorbed before reaching the caecum of birds (Han, Castanon,
sources (DLM-­99% and HMTBA-­88%). Meanwhile, Viana et al. (2009) Parsons, & Baker, 1990). This factor corroborates the difference found
studied two dietary methionine sources (DLM-­99% and HMTBA-­88%) in the sources because the quails fed the DLM-­99% source showed
observed no significant difference in feed intake. better performance than the quails fed diets with the HMTBA-­88% or
The same pattern of feed intake was observed for egg produc- HMTBA-­84% sources.
tion and weight. The diet without supplementation caused worsened Conversely, Han et al. (1990), Dibner, Atwell, and Ivey (1992)
poultry performance because methionine is crucial for egg production and Richards, Atwell, Vázquez-­Añón, and Dibner (2005) found that
and weight (Costa et al., 2009; Reis et al., 2011). The other variables, HMTBA-­88% was absorbed throughout the entire intestine, and this
including egg mass and feed conversion per egg mass and feed con- source may also be absorbed actively (Dibner et al., 1992) and as rap-
version per dozen eggs, showed the same pattern because they are idly as the l-­methionine form (Knight & Dibner, 1984), corroborating
calculated based on egg production and egg weight and feed intake. the findings of Larbier (1988) and Han et al. (1990), who observed
There was no difference in feed intake, egg weight or egg mass 100% and 98% absorption efficacy for DLM-­99% and HMTBA-­88%,
between the two alternative sources (HMTBA-­88% vs HMTBA-­84%); respectively, concluding that bioefficacy is not related to absorption.
however, there was a difference in the egg production and feed con- Another factor that may favour the increased efficacy of DLM-­
versions measured (feed conversion per egg mass—FCEM, and feed 99% lies in the fact that HMTBA has few monomeric fractions, result-
conversion per egg dozen—FCDZ), with the quails fed the diet with ing in its low bioefficacy (Saunderson, 1991; Van Weerden, Schuttle,
HMTBA-­88% showing improved performance in these variables. & Bertram, 1992). However, this idea was refuted by Martín-Venegas,
A significant linear effect of the levels of supplementation with di- Geraert, and Ferrer (2006) when comparing the in vivo and in vitro ab-
gestible Met + Cys on the three supplemental sources was observed for sorption of HMTBA containing only monomeric fractions.
all variables (Table 2), that is, increased dietary supplementation with Table 3 shows that the dietary methionine sources used (DLM-­
methionine + cystine promoted lower feed intake, higher egg produc- 99%; HMTBA-­88% and HMTBA-­84%) affected the relative shell
tion, egg weight and egg mass and lower feed conversion per egg and weight (p = .0001), relative albumen weight (p = .0001), relative yolk
per egg dozen. However, when comparing the alternative sources, di- weight (p = .0001) and specific gravity of eggs (p = .0142). The shell
etary supplementation with digestible Met + Cys using HMTBA-­88% thickness (p = .899) was not affected by the methionine source.
promotes improved performance of laying Japanese quails. The analysis of orthogonal contrasts for the significant variables
Table 2 shows that there is a difference in methionine utilisation (relative weight of shell, albumen and yolk and specific gravity) showed
efficacy depending on the alternative supplemental sources, with an that animals fed the control diet laid eggs with lower absolute values
81% mean bioefficacy assessed for the HMTBA-­88% source and 79% for shell and albumen weight and were more prone to having a thin
for the HMTBA-­84% source. shell based on specific gravity analysis. These results show the impor-
The difference found in the bioefficacy of the supplemental me- tance of the supplemental dietary methionine source for laying quails
thionine sources could result from their composition and the form of because methionine is the first limiting amino acid for laying poultry
absorption of their constituents because supplemental methionine (Costa et al., 2009; Reis et al., 2011), especially for shell thickness, as
sources (HMTBA) have analogues that differ from methionine from this variable is key for egg integrity.
standard sources. The analogues have a hydroxyl group (OH) instead The comparison between methionine sources and their effects
of the amine group (NH 2), located in the alpha-­carbon of the molecule. on relative shell weight, relative albumen weight, relative yolk weight
8 | VIEIRA et al.

and specific gravity indicates that diets with DLM-­99% led to eggs (Gonzales-­Esquerra et al., 2007; Vazquez-Anon, Kratzer et al., 2006;
with more albumen, and less shell and yolk, and no difference was Vazquez-Anon, Gonzalez-Esquerra et al., 2006).
observed for this variable between the alternative sources. The alter-
native sources had no effect on weight shell or specific gravity and are
therefore equivalent. 4 | CONCLUSION
A significant linear effect (Table 3) of the levels of supplementa-
tion with digestible Met + Cys from the DLM-­99% source was ob- The bioefficacy values of the HMTBA-­88% and HMTBA-­84% sources
served on the variables relative yolk weight, relative shell weight and compared to the DLM-­99% source on an equimolar basis were 81 and
relative albumen weight and specific gravity, but no difference about 79%, respectively, for the performance variables, and 83 and 74 for
shell thickness. Only shell thickness not showed linear effect from the the variables related to egg quality.
HMTBA-­88% source. Was observed similar effect for the HMTBA-
84% source, in relation the HMTBA-88%, except to specific gravity,
that no linear effect was observed for HMTBA-84% source. Table 3 O RC I D
shows the occurrence of equivalence in the use of the supplemental
D. V. G. Vieira http://orcid.org/0000-0002-7407-9597
methionine sources for all variables, except relative yolk weight and
M. R. Lima http://orcid.org/0000-0002-9897-6209
relative weight albumen. A mean bioefficacy of 83% was observed for
the HMTBA-­88% source and 74% for the HMTBA-­84% source.
There are several controversies regarding the results about the
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