Dynamics of thymol dietary supplementation in quail (Coturnix japonica): Linking bioavailability, effects on egg yolk total fatty acids and performance traits
Dynamics of thymol dietary supplementation in quail (Coturnix japonica): Linking bioavailability, effects on egg yolk total fatty acids and performance traits
Maria E. FernandezID 0 1 2
Jackelyn M. KembroID 0 1 2
Maria L. BallesterosID 1 2
Jorge M. Caliva 0 1 2
Raul H. Marin 0 1 2
Maria C. LabaqueID 0 1 2
0 Consejo Nacional de Investigaciones Cient ??ficas y T e ?cnicas (CONICET), Instituto de Investigaciones Biol o ?gicas y Tecnol o ?gicas (IIByT), C o ?rdoba, Argentina, 2 Universidad Nacional de C o ?rdoba, Facultad de Ciencias Exactas , F ??sicas y Naturales , Instituto de Ciencia y Tecnolog ??a de los Alimentos (ICTA), C o ?rdoba, Argentina, 3 Universidad Nacional de C o ?rdoba, Facultad de Ciencias Exactas, F ??sicas y Naturales, C a ?tedra de Qu ??mica Biol o ?gica , C o ?rdoba, Argentina, 4 Consejo Nacional de Investigaciones Cient ??ficas y T e ?cnicas (CONICET) , Instituto de Diversidad y Ecolog ??a Animal (IDEA), C o ?rdoba, Argentina, 5 Universidad Nacional de C o ?rdoba, Facultad de Ciencias Exactas, F ??sicas y Naturales, C a ?tedra de Diversidad Animal II, C o ?rdoba, Argentina, 6 Universidad Nacional de C o ?rdoba, Facultad de Ciencias Exactas , F ??sicas y Naturales, C a ?tedra de Ecolog ??a, C o ?rdoba , Argentina
1 Editor: Juan J. Loor, University of Illinois , UNITED STATES
2 Funding: This research was supported by Argentine grants from FONCyT (Fondo para la Investigacio ?n Cient ??fica y Tecnolo ?gica) grant number PICT 2014-2764 , awarded to RHM, MCL and JMK, website
Phytogenic additives such as thymol are encountering growing interest in the poultry industry. However, there are still questions concerning dynamics of their bioavailability, biological action, optimal dosage and duration of supplementation needed to achieve meaningful effects, as well as persistence of induced changes after supplement withdrawal. We studied the link between the dynamics of free thymol concentration and the changes in fatty acids composition in quail egg yolk, both during a month-long chronic dietary supplementation and after 3 weeks of supplement withdrawal (post-supplementation). Fifty, 85 days-old, female quail of homogeneous body weights (251?1g) in egg-laying peak were used. To evaluate potential dose-dependent effects, three increasing doses 2, 4, and 6.25 g of thymol/kg of feed (THY2, THY4 and THY6, respectively) and two controls were evaluated (n = 10). In parallel, we assessed the concomitant changes in free thymol excretion, potential liver histopathological changes, and birds? performance traits. Egg yolk and droppings show a dose-dependent increase in THY concentration after 9 days of supplementation and a decrease after post-supplementation. In egg yolk, these changes were accompanied by reduced saturated fatty acid concentrations achieved by 28 days of supplementation in
THY2 and 14 days of supplementation in THY4 and THY6. However, after
post-supplementation the aforementioned effect disappeared in THY2 but not in THY4 and THY6. While
THY2 failed to increase polyunsaturated fatty acids, THY4 and THY6 increased
polyunsaturated fatty acids by day 14 of supplementation and remained increased after
post-supplementation. Fatty acids changes induced by thymol are consistent with improved nutritional
quality of eggs. No treatment effects were observed in liver histopathology and female
performance. Findings suggest that both dose of thymol and duration of supplementation
33820180100125CB, awarded to MCL, website
Competing interests: The authors have declared
that no competing interests exist.
modulate thymol and fatty acids concentrations in egg yolk and thymol concentration in
droppings. Furthermore, the persistence of those effects after post-supplementation period
is also a dose-dependent phenomenon.
Phytogenic additives are encountering growing interest in poultry nutrition due to their
potential beneficial influence on lipid metabolism, useful gut microflora, nutrient absorption,
performance, health and welfare [
]. In particular, dietary supplementation with thymol (THY; 2
isopropyl-5-methylphenol) or mixtures that contain THY, has been proposed as strategy to
enhance poultry productivity in parameters such as growth, feed conversion, egg laying rate and
egg physico-chemical quality [
]. Additionally, THY potential for modulating the fatty acid
(FA) profile and preventing lipid peroxidation in certain tissues of a variety of species [
promoted research aimed at improving nutritional quality of meat [
] and egg products
]. From a human nutritional standpoint, consuming products with higher levels of n-3
polyunsaturated FA (n-3 PUFA) and lower saturated FA (SFA) is associated with a number of
physiological and health-beneficial effects, such as prevention of some chronic diseases [
Also, in the context of early avian nutrition, adequate amounts of PUFA in the yolk are essential
to meet the demands of the developing embryo [
]. Modulation of the FA profile by diverse
dietary supplements containing THY has been largely attributed to THY antioxidant activity via
scavenging of free radicals, enhancing the bird?s endogenous enzymatic and non-enzymatic
antioxidants, chelating of metal ions and regulation of different signaling pathways [
As a bioactive compound, THY has potential for physiological effects at various levels [
vitro results in different cell lines and experimental systems showed that THY promotes
doseresponse effects (reviewed in [
]). However, in vivo studies in poultry, reflect inconsistencies in
relation to dose-response effects, which can be explained, in part, by substantial differences in
supplementation protocols used [
]. In particular, there are fundamental differences
regarding presentation form of the supplement (ranging from herbs to commercial mixtures
with several EO), doses administered (<mg to g/kg feed), duration of supplementation (days to
months) and age of animals supplemented (chicks to adults), which could ultimately impact,
among other aspects, on bioavailability or amount of the supplement that can be absorbed, used
and/or stored by the animal [
]. Although the contributions of these studies are of great
importance, the wide range of supplementation protocols hinders an adequate understanding of
the scope of THY effects, as well as the characteristics of its in vivo mode of action, thus, making
it difficult to identify supplementation aspects (e.g. absorption, target tissues, etc.) that are
important in order to promote accurate nutritional recommendations. This background
highlights the need for studies using pure THY (as a single compound), at various doses throughout
the supplementation protocol, to determine whether there are doses or durations of
supplementation thresholds capable of promoting biologically relevant responses.
In species such as rabbits, pigs, rodents and humans there is a fairly good number of studies
on THY absorption, digestion, metabolism, excretion and bioavailability, which provide
valuable background information in the context of the nutrition of monogastric animals in general.
However, these studies have been conducted in acute administration and provided short-term
data regarding the permanence of these compounds and/or their derivatives in the animal
]. On the contrary, as stated previously, application protocols proposed in animal
nutrition involve chronic supplementation of these compounds. In this regard, few studies in
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poultry evaluated the bioavailability or concentration of supplemented compounds in target
tissues. The scarceness of this information has been attributed partially to methodological
difficulties. Recently, methodologies based on HS-SPME coupled to GC-MS and SPE coupled to
UHPLC-QTOF-MS have been developed, enabling these determinations [
]. Of special
interest for us are the results in broiler chickens  demonstrating that THY from thyme
herb (consisting of leaves and flowers without stems) at 0, 1, 2, 3 and 10 g/kg of feed is
efficiently absorbed, distributed to various tissues and excreted after a supplementation trial of 35
days. They also observed an increased intestinal THY concentrations in the group with 10g/kg
feed compared to the other groups. Likewise, it has been demonstrated that THY
concentration in the egg yolk reached a plateau after 12 days of dietary supplementation, in laying hens
fed with thyme extracts at doses equivalent to 2.24 and 3.36 g of THY/kg feed [
Additionally, they observed that once supplementation was withdrawn, THY concentration of the yolk
decreased gradually. However, these studies, as mentioned above, not only use supplements
that are mixtures of different compounds, but also they lack a joint assessment of the dynamics
of changes in bioavailability and biological effects (i.e., feed intake, body weight, egg
physicochemical characteristics, etc.) potentially promoted during supplementation (S) or in the
postsupplementation (pS) period. This approach is key to understand how supplemented
compounds modulate beneficial effects on animal body, thus contributing to determine to what
extent the biological effects depend on the circulating levels and/or the concentration of the
active compounds in target tissues [
]. Measuring bioavailability may also be relevant in
context of the safety of edible animal by-products (i.e., eggs and meat).
Although THY has the ?generally recognized as safe? (GRAS) status of US
governmentapproved food additives, with safety levels calculated in a wide range of animal species by the
European Food Safety Authority [
], further studies complementing toxicological and
persistence effects after chronic supplementation in vivo are still needed. Moreover, recent reviews
have also call for the need of detailed assessment of toxic effects on liver, due to its role in
phenolic compounds? metabolism, as xenobiotic biotransformer and in circulatory system [
Thus, we studied the link between the dynamics of free THY concentration and the changes
in FA composition in quail egg yolk, both during a month-long chronic dietary
supplementation and after 3 weeks of supplement withdrawal. To evaluate whether the observed effects are
dose dependent, 3 increasing doses of THY (2, 4, and 6.25 g of THY/kg of feed) were evaluated.
In parallel, we assessed the concomitant changes in free THY excretion as a non-invasive
indicator of the presence of THY in circulation. We also monitored potential liver
histopathological changes due to toxicity, as well as female performance traits.
Materials and methods
All experimental procedures were in compliance with the Guide for the Care and Use of
Laboratory Animals issued by the National Institute of Health [
]. The experimental protocol was
approved by the Institutional Committee for the Care and Use of Laboratory Animals (Comite?
Institucional para el Cuidado y Uso de Animales de Laboratorio (CICUAL) of the Facultad de
Ciencias Exactas, F??sicas y Naturales, Universidad Nacional de Co?rdoba (ACTA 4/2015
Resolucio?n 571-HCD-2014). The raw data of all variables presented herein is publicly available on
figshare, doi: 10.6084/m9.figshare.7589066.
Animals and husbandry
Adult female Japanese quail (Coturnix japonica) taken from a population of a single 230-bird,
hatched in our laboratory, were used in this study. Husbandry was performed according to
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laboratory routines described elsewhere [
]. Briefly, at 28 days of age, females were
individually housed in cages measuring 20cm ? 45cm ? 25cm (length ? width ? height), allowing
them to establish visual and auditory contact with each other, while permitting individual
measurements of feed consumption and egg production. An individual feeder and an
automatic nipple drinker were positioned in each cage. At all stages feed and water were provided
ad libitum. The photoperiod was 14h light and 10h dark (0600?2000h; approximately 300?320
lx). The environmental temperature was maintained at 24 ? 2?C.
A total of 50 eighty-five days-old female quail of homogeneous body weights (251 ? 1 g;
Mean ? SE) and in their egg-laying peak were selected from an initial group of 96 female
individuals. All birds continued receiving a basal diet between 85 and 100 days of age and all
variables (see below for details) were registered along this period (Pre-supplementation period, Fig
1). From 100 to 128 days of age (S period, Fig 1), females were randomly assigned to either a
chronic dietary supplementation with one of 3 THY doses (THY2, THY4 and THY6; 10 females
each) or to one of 2 control groups (basal diet, BASAL, and basal diet with vehicle solution,
VEHICLE; 10 females each). Once finished the S period, half of the quail were slaughtered for
histological analysis (n = 25; Slaughter 1, Fig 1) and the other half were subjected to a pS period
of 21 days, during which all females received the BASAL diet (pS, Fig 1). After this period, the
remaining 25 animals were also slaughtered for histological analysis (Slaughter 2, Fig 1). In this
way, we assessed whether THY induced changes in variables indicative of tissue damage and if
so, whether the potential damages could reverse after 3 weeks of supplement withdrawal.
To assess changes in egg yolk total FA composition, egg physical characteristics, laying rate,
body weight, feed intake and general welfare, during the S and pS periods, five sampling points
were defined (Sample points, Fig 1): one initial sample point (at 0 days of S); three sample
points during the S period (at 7, 14 and 28 days of S); and one sample point at the end of the
pS period (21 days of pS). It should be noted that for determinations of THY concentration,
Fig 1. Timeline scheme. The age of the birds (days) is indicated on the horizontal axis. All birds received the basal diet
between 85 and 100 days of age. From 100 to 128 days of age, females were subjected to chronic dietary
supplementation (S). Five diets (N = 50; n = 10) were administrated (indicated with boxes): two controls (BASAL and
VEHICLE) and three increasing doses of THY (THY2, THY4 and THY6, corresponding to 2, 4 and 6.25 g of THY/kg
feed, respectively). Vertical dotted lines indicate changes in the diet of each experimental group. Once finished the
supplementation period, half of the quail were slaughtered for histological analysis (Slaughter 1). The other half of the
quail was subjected to a post-supplementation period of 21 days (pS) during which the basal diet was reestablished.
Finished de pS period, the remaining birds were slaughtered (Slaughter 2). To assess dynamics five sample points were
defined: one initial sample point; three sample points along the S period and one at the end of the pS period.
4 / 23
eggs and quail droppings were collected offset from the other variables measured by 2 days,
thus were collected on days 2, 9, 16, and 26 days of S, and 17 days of pS, respectively.
Thus, a bi-factorial design combining the effects of the diet supplied (five levels) and time
of sampling (five levels) was established for the variables studied.
Thymol was commercially obtained from Sigma-Aldrich (SAFC1, 99%; FCC, Saint Louis,
MO, USA). Supplemented quail were provided 2, 4 or 6.25 g of THY per kg of feed. For this
end, THY was prepared in a 2.4, 4.8 and 7.5% w/v ethanolic solution and uniformly sprayed
on fresh feed [
]. The feed was prepared and administrated daily, to minimize THY
volatilization from feed before its consumption by females. The doses were selected considering two
factors. The first, the concentration range for which the transfer of THY to the chicken and/or
quail egg has been established [
]. Second, the concentrations of THY supplementation
that have shown biological effects. For example, it was shown that THY2 reduces female quail
fear responses when birds are exposed to stressful situations without affecting the bird?s
locomotor activity ; and that THY6 enhances PUFA deposition to the detriment of SFA in yolk
total fatty acids and triglycerides of the quail fertile egg [
]. In this context, THY4 is and
intermediate dose between the last two. Nutrient and FA compositions of the diets supplied were
identical between each other and are reported in S1, S2 and S3 Tables, and are in accordance
with the National Research Council recommendation [
Thymol quantification in egg yolk, quail droppings and feed
Egg yolks and droppings of each female were obtained at each sample point (Sample points,
Fig 1) and THY concentration was immediately measured by head space-solid phase
microextraction followed by gas chromatography-mass spectrometry (HS-SPME/GC-MS) according
to Fernandez et al. [
]. SPME was performed with a manual holder with 100 um
polydimethylsiloxane (PDMS) fiber. An aliquot of egg yolk (3 g), droppings (5 g) or feed (5 g) was
put in a sealed 20 mL glass vial, spiked at the corresponding level with the standard m-cresol
(Sigma-Aldrich; 99% (GC); Saint Louis, MO, USA) stock solution, and vortexed for 5 min.
The vials were placed in a water bath and the PDMS fiber was exposed to the headspace for 30
min at 60?C and 5 min at 40?C for egg yolk and droppings or feed, respectively. The fiber was
then inserted directly into the GC injector for desorption at 250?C for 10 min in splitless
mode. Chromatography analysis was carried out in a Perkin Elmer Clarus 600 equipped with a
PSSI injector and a quadrupole MS detector (Perkin Elmer, USA). Turbo Mass 5.4.2 software
was used to control and acquire data from GC?MS. All the separations were performed using
a Perkin Elmer fused silica DB 5 MS capillary column (60 m, 0.25 mm ID, 0.25 um film
thickness), with High-purity helium (99.998%) as a carrier gas (49.6 psi). The splitless injection
mode was selected. Electron-impact Ionization was carried out in the mass spectrometer
under vacuum with 70-eV ionization energy. Samples were analyzed under the following
chromatographic and MS detection conditions: initial oven temperature was set at 100?C (held for
2 min), and then raised to 230 at 10?C/min rate. A column head pressure of 14.99 psi and an
injector temperature of 280?C were set. The GC transfer line was maintained at 250?C. The
fiber was desorbed in the GC injector port for 10 min. Chromatograms were acquired in scan
mode, which scans the quadrupole from m/z = 50 to m/z = 300 (scan time: 0.20 s, inter-scan
time: 0.10 s). All quantitative analyses were performed in TIC mode. The compounds were
identified by comparing their mass spectra with those of the libraries of the NIST MS search
2.0. The main components were further identified by co-injection of commercial standards.
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Total fatty acid analysis in egg yolks
Eggs were obtained at each sample point (Sample points, Fig 1) and stored at -20?C until
yolk total fatty acids analysis. Lipids were extracted from the yolk following homogenization
in a suitable excess of chloroform/methanol (2:1 v/v) [
]. The solvents were removed
under reduced pressure in a rotary evaporator. Lipids were subjected to alkaline
saponification (1 mol/L potassium hydroxide in methanol), and the unsaponifiable matter was
extracted with n-hexane. The fatty acid methyl esters (FAMEs) were prepared by
transmethylation through treatment with 1 mol/L sulfuric acid in methanol and analyzed by gas
chromatography/mass spectrometry (GC/MS) [
]. All chemicals used in this study were
reagent-grade commercial products. FAMEs were analyzed by gas chromatography on a 60
m fused capillary column with an internal diameter of 0.25 mm (PerkinElmer Elite-WAX
Polyethylene Glycol). The analysis was performed on a PerkinElmer Clarus1 600 GC/MS
system equipped with a flame ionization detector (Waltham, MA, USA). Helium was used
as carrier gas (constant flow of 49.6 psi). The injection port temperature was 250?C and the
detector temperature was 250?C. The oven temperature was initially held at 180?C for 5
min, then increased at 4?C/min to 200?C and held for 5 min and finally increased at 3?C/
min to 230?C and held for 25 min. Peak identification was carried out by comparing the
known retention times for the fatty acids reported with the temperature program and the
chromatographic system used. A solution of known concentration of nonadecanoic acid
methyl ester (Sigma Aldrich, 98.0% (GC); Saint Louis, MO, USA) as was used internal
standard to estimate the content of each fatty acid in the sample.
Female performance traits
Female performance was evaluated through the measurement of body weight, feed intake,
egg laying rate, egg physical characteristics and a general welfare quality assessment. Body
weight was measured once a week throughout the experimental period. Daily feed intake
(DFI) was estimated as the difference between the amount of feed supplied to each animal
(60 g) and the rest that remained in the feeders the next day (g of feed/day/quail) [
Egg laying was registered daily and the weekly laying rate was calculated as: (number of
eggs / 7 days) x 100. Egg weight, quality characteristics (such as intact, membranous, soft
shell and broken shell), morphometric characteristics (egg shape index = (egg width / egg
length) x 100) and percentage of constituents (egg yolk, albumen and shell) were registered
at each sampling point [
A welfare assessment based on observations of physical characteristics was made. Female
skin lesions and plumage status were evaluated following a procedure proposed by
Pellegrini et al. [
] that is an adapted version of the protocol developed by the Welfare Quality
consortium . Briefly, skin lesions, which include wounds that have not healed in the
legs, rear end, chest, cloacae and wings were determined using a score scale from 0 to 2,
where "0" represents no lesions or scratches, "1" represents at least one lesion < 0.5 cm
diameter or less than 3 pecks (punctiform damage ~ 0.1 cm of diameter) or scratches, and
"2" reflects one lesion 0.5 cm of diameter or more than 3 pecks or scratches. Plumage
damage was also determined using a score scale from 0 to 2 as follows: "0" represents
individuals with no plumage damage or slight wear (only single feathers lacking), "1" represent
individuals with one or more body parts that have moderate wear (i.e. damaged feathers
worn or deformed) or one or more featherless areas < 1.5 cm in diameter at the larger
extent and "2" corresponded to individuals that have at least one featherless area > 1.5 cm
in diameter at the largest extent. Foot pad dermatitis, for which both feet were analyzed and
the foot with the worst condition was scored according to the following: "0" representing
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feet intact, no or minimal proliferation of epithelium, "1" corresponded to necrosis or
proliferation of epithelium or chronic bumble foot with no or moderate swelling, and "2"
indicated swollen dorsally visible. Eye pathologies, which include swelling of the eyelids and the
skin around the eyes, closure of the eye/eyes and discharge from the eyes were classified as
"0" when no evidence of eye pathologies were observed or "1" if a there were eye pathologies.
Additionally, dirt from the legs, cloacae and belly was examined for signs of diarrhea
potentially caused by dietary supplementation.
Liver histological analysis
At each slaughter point birds were euthanized by decapitation and livers were removed and
stored in buffered formalin at 10%, processed routinely and stained with hematoxylin and
eosin (H&E) and periodic acid-Schiff (PAS). Each slide was examined blinded, with a light
microscope (Olympus X-785) and photographed with a digital camera (Moticam Camera
2300, 3 Megapixels). To evaluate histological alterations of liver, tissue was divided into 8
random equal areas to ensure there was no overlapping of the studied areas. For each area,
the extension and number of each alteration were recorded with a microscope at 40X
magnitude. Histopathological index of liver (HIliv) were estimated using a semi-quantitative
protocol following [
], modified by our colleagues [
]. Briefly, alterations were classified
into four major reaction patterns each one of those including alterations that concern to
different functional units of the liver or to the whole organ: RP1, circulatory disturbances
(dilatation of sinusoids, vascular congestion, hemorrhage); RP2, regressive changes
(steatosis or fatty degeneration, hydropic degeneration, nuclear alteration, fibrosis, necrosis); RP3,
progressive changes (oval cells, cell hypertrophy and hyperplasia); and RP4, liver
inflammation (leukocyte infiltration). Then, for each RP, an index was calculated based on two
factors: the pathological importance of the lesions (importance factor, W) (range 1?3) and the
extension of pathological change (score value, a) from 0 (unchanged) to 8 (extreme
occurrence). Finally, a total liver histopathological index (HIliv) was calculated by adding the
single RP liver indices of each individual quail. A greater value of (HIliv) reflects the most
severely affected individual.
General and generalized linear mixed models were used in order to analyze the effects of the
diet supplied (5 levels of factor) and time of sampling (5 levels of factor) throughout
experimental manipulation. The diet supplied and time of sampling were included as fixed effects
and female identity was included as a random effect for THY concentration, FA
composition, feed intake, body weight, morphometric variables and percentage of egg constituents.
For histological analysis, a two-way ANOVA including the diet supplied and the slaughter
batch (28 days of S or 21 days of pS) as effects was used. Egg-laying was analyzed as
cumulative number of eggs using a one-way ANOVA with the diet supplied as unique factor. Data
were analyzed according to normal distribution unless stated otherwise and assumptions of
the tests were verified. The variables THY concentration, weekly body weight, SFA,
arachidonic, eicosapentaenoic and docosapentaenoic acids of egg yolk were transformed before
analysis (log10 was used for THY concentration in the yolk and weekly body weight, root
square for THY concentration in the droppings, and Logit for all other variables). Palmitic
and stearic acids and egg weight that were analyzed according to gamma distribution. A
Pvalue of <0.05 was considered to represent significant differences. DGC test was used for
post hoc comparisons. All statistical analyses were performed with ?R? (The R Foundation
for Statistical Computing) through a user-friendly interface implemented in InfoStat [
7 / 23
Thymol quantification in egg yolks and droppings
The dose-dependent effects of the diet supplied over the time of sampling on THY
concentration in the egg yolk and droppings are shown in Fig 2. In the egg yolk, significant effects
(Table 1 and Fig 2A) of both the diet supplied and time of sampling on THY concentration
were found. Post-hoc analysis showed that THY concentration increased on 9 days of S.
Subsequently, the concentration reached a plateau that lasted until the end of the S period. After
supplement withdrawal, THY concentration decreased. Regarding the THY doses tested, it was
found that the higher the dose supplemented, the higher the concentration it is detected. In
dropping samples, a significant interaction (Table 1 and Fig 2B) between the diet supplied and
time of sampling was observed, with the same general pattern described in the egg yolk with
the exception that THY6 was higher than THY4 only at 16 and 26 days of S and that THY2
was higher at 26 days than at 16 days of S.
Changes in egg yolk total fatty acid composition
The dose-dependent effects of the diet supplied over the time of sampling on egg yolk FA
concentration are shown in Figs 3?6. Significant effects (Table 1) of the diet supplied, time of
sampling and their interaction was found for SFA and PUFA. As expected, post-hoc analysis
showed that control (BASAL and VEHICLE) females maintained constant SFA and PUFA
values throughout experimental manipulation (Fig 3A?3C). On the contrary, along the S period
THY4 and THY6 induced PUFA increments that remained enhanced even at 21 days of pS,
while THY2 failed to increase PUFA (Fig 3C). Specifically, all THY supplemented diets
increased 22:6 and 20:5 from 7 days of S on, but only THY4 and THY6 showed higher
concentrations of 18:3 and 20:4, from 14 days of S onwards (Fig 6B?6E). In addition, THY2 and
THY4 required 14 days of S for increasing 22:5, while THY6 needed only 7 days of S for
achieving similar changes (Fig 6F). On the other hand, THY2 required 28 days of S to reduce
the SFA concentration, while THY4 and THY6 only needed 14 days of S for a reduction of
similar and even greater magnitude, respectively. However, after 21 days of pS the
aforementioned effect disappeared in THY2 but not in THY4 and THY6 (Fig 3A). Concomitantly,
reduced 16:0 and 18:0 concentrations were achieved by 14 days of S in THY4 and THY6 and
by 28 days of S in THY2. After 21 days of pS those effects continued in all diets, except for the
16:0 and 18:0 concentrations in THY2 and THY6, respectively, that returned to initial levels
(Fig 4A and 4B). No differences were observed between the diet supplied, time of sampling or
their interaction in MUFA (Fig 3B). However, a transient decrease of 16:1 was observed by 7
and 14 days of S in THY2 (Fig 5A and 5B) and at 21 days of pS in THY4 and THY6 respect to
their diet and time counterparts.
Female performance traits
The effects of the diet supplied over the time of sampling on variables related to performance
(body weight, feed intake, egg laying, morphometric characteristics and constituents of eggs)
are shown in Table 2. Because no effects of the diet supplied, time of sampling or interaction
were found, the Table is only showing the overall averages. Considering that no differences in
the daily feed intake was observed between treatments (30.3 ? 1 g/day/animal), it can be
assumed that the amount of THY consumed was proportional to the dose supplemented. By
measures of THY concentration in the feed (S4 and S5 Tables) we were able to estimate a daily
THY intake of 0.26, 0.70 and 1.2 mg/day/animal for THY2, THY4 and THY6, respectively.
8 / 23
Fig 2. Thymol concentration in quail egg yolk and droppings. Mean ? SEM of thymol concentration (ng/g) in the
(A) egg yolk and (B) droppings from females fed on diets with 2, 4, and 6.25 g of thymol/kg of feed (THY2, THY4 and
THY6, respectively) at 2, 9, 16, and 26 days of the supplementation period (S) and after 17 days of supplement
withdrawal (pS). Arrow pointing down indicates the end of the S period. In Fig 2A different letters (a, b, c) and
numbers (1, 2, 3) indicate statistical differences for the diet supplied and time of sampling, respectively. In Fig 2B
different letters (a, b, c, d, e, f) indicate statistical differences for the interaction. The same 15 females (5 per THY
supplemented diet) were sampled on each of the five sample points (75 eggs and 45 droppings samples total). BASAL
and VEHICLE samples were evaluated at the beginning and end of the day at each sample point to assure the absence
Thus, concentrations of THY transferred to the egg yolk and droppings during the plateau
respectively correspond to ~0.13?0.25% and ~1.57?3.47% of the amount THY ingested.
9 / 23
Regarding general welfare, 100% of the birds of all treatments showed optimal plumage on
the back and rump and around the cloacae, no skin lesions were observed, and only minimal
proliferation of epithelium were observed (category 0) throughout S and pS periods. In
relation to plumage of the neck and head, eye pathologies and dirt in the cloacae and legs, only a
few isolated cases of category 1 were observed and were not associated with any particular
Liver histological analysis
As a general description of the state of experimental animals, representative photomicrographs
and the frequency of histopathological alterations registered in all livers analyzed regardless of
the diet supplied or sample point are presented in Figs 7 and 8, respectively. The presence of
steatosis or fatty degeneration was recorded in 89% of the livers evaluated (Figs 7B and 8).
Sinusoidal dilatation and vascular congestion (both considered reversible changes) were
observed respectively in 35% and 26% of the samples (Figs 7C and 8). A low number of small
10 / 23
Fig 3. Concentration of saturated, monounsaturated and polyunsaturated fatty acids of quail egg yolk. Mean ? SEM of content (g/100 g FAME) of (A) saturated
(myristic + pentadecanoic + palmitic + stearic acids), (B) monounsaturated (palmitoleic + oleic acids) and (C) polyunsaturated (linoleic + linolenic + arachidonic
+ docosahexaenoic + eicosapentaenoic + docosapentaenoic acids) fatty acids of quail egg yolk laid by females fed on the control diets (BASAL and VEHICLE) or
supplemented with 2, 4, and 6.25 g of thymol/kg of feed (THY2, THY4 and THY6, respectively) at 0, 7, 14, and 28 days of the supplementation period (S) and after 21 days
of supplement withdrawal (pS). Arrow pointing down indicates the end of the S period. Brackets refer to measurements within a time of sampling. Different letters (a, b, c,
d) indicate statistical differences. The same 25 females (5 per diet supplied) were analyzed on each of the five sample points (125 eggs total).
necrotic foci (12%) and foci of oval cells (associated with tissue regeneration) were found (Figs
7D and 8). Both the leukocyte infiltration and the pyknotic nuclei were recorded at low
frequency (<5%). Thus, among histopathological alterations, regressive changes (PR2: steatosis
or fatty degeneration and necrosis) registered the highest frequency, followed by circulatory
alterations (PR1: sinusoidal dilatation and vascular congestion) and finally progressive and
inflammatory changes (PR3: foci of oval cells and PR4: inflammation) (Fig 8).
The effects of dietary supplementation on liver histopathological indices of animals that
had gone through 28 days of S and the pS period are shown in Table 3. Because no treatment
effects were found, only overall averages are shown. Moreover, as histopathological alterations
corresponding to Rp3 and Rp4 were observed only in a few individuals that were not
associated with any particular treatment, p-values in those cases were not calculated. Total
histopathological indices varied between 17.6 and 27.6.
11 / 23
Fig 4. Concentration of saturated fatty acids of quail egg yolk. Mean ? SEM of content (g/100 g FAME) of (A) palmitic (16:0) and (B) stearic acids (18:0) of quail egg
yolk laid by females fed on the control diets (BASAL and VEHICLE) or supplemented with 2, 4, and 6.25 g of thymol/kg of feed (THY2, THY4 and THY6, respectively) at
0, 7, 14, and 28 days of the supplementation period (S) and after 21 days of supplement withdrawal (pS). Arrow pointing down indicates the end of the S period. Different
letters (a, b, c) indicate statistical differences. Brackets refer to measurements within a time of sampling. The same 25 females (5 per diet supplied) were analyzed on each of
the five sample points (125 eggs total).
The main contribution of the present work is to show that both the dose of THY and the
duration of the S period directly modulate the FA concentrations in quail eggs showing positive
Fig 5. Concentration of monounsaturated fatty acids of quail egg yolk. Mean ? SEM of content (g/100 g FAME) of (A) palmitoleic (16:1) and (B) oleic acids (18:1) of
quail egg yolk laid by females fed on the control diets (BASAL and VEHICLE) or supplemented with 2, 4, and 6.25 g of thymol/kg of feed (THY2, THY4 and THY6,
respectively) at 0, 7, 14, and 28 days of the supplementation period (S) and after 21 days of supplement withdrawal (pS). Arrow pointing down indicates the end of the S
period. Different letters (a, b) indicate statistical differences. Brackets refer to measurements within a time of sampling. The same 25 females (5 per diet supplied) were
analyzed on each of the five sample points (125 eggs total).
12 / 23
Fig 6. Concentration polyunsaturated fatty acids of quail egg yolk. Mean ? SEM of content (g/100 g FAME) of (A) linoleic (18:2), (B) linolenic
(18:3), (C) arachidonic (20:4), (D) docosahexaenoic (22:6), (E) eicosapentaenoic (20:5) and (F) docosapentaenoic (22:5) acids of quail egg yolk laid
by females fed on the control diets (BASAL and VEHICLE) or supplemented with 2, 4, and 6.25 g of thymol/kg of feed (THY2, THY4 and THY6,
respectively) at 0, 7, 14, and 28 days of the supplementation period (S) and after 21 days of supplement withdrawal (pS). Arrow pointing down
indicates the end of the S period. Different letters (a, b) indicate statistical differences. Brackets refer to measurements within a time of sampling.
The same 25 females (5 per diet supplied) were analyzed on each of the five sample points (125 eggs total).
13 / 23
Considering that no effects of the diet supplied, time of sampling or their interaction were found, only overall means are shown. Two control (BASAL and VEHICLE)
and three supplemented diets (2, 4, and 6.25 g of thymol/kg of feed) were used. The five sampling time points used were at 0, 7, 14, and 28 days of the supplementation
period and after 21 days of supplement withdrawal.
Egg shape index = (egg width / egg length) x 100
changes that remain apparent even after 21 days of THY withdrawal. Interestingly, the
phenomenon was observed without changes in liver histology or impairments in any of the female
productive traits registered. Furthermore, the bioavailability of THY both in the egg yolk and
in droppings (i.e. an indicator of changes in circulating levels of THY) was also directly related
to the concentration of THY and the duration of the supplementation.
In general, THY supplementation induced increments in PUFA concentration with a
decrement in SFA in the egg yolk that were evident after 14 days of supplementation. These
changes in the FA profile are consistent with an improved nutritional quality of eggs both
from human nutrition standpoint (i.e. for consumption of healthy eggs; [
]) and for early
avian nutrition and hatching success [
By simultaneously assessing the dynamics of changes in THY and FA concentration along S
and pS periods in several doses of THY, we were able to display the relations between the
pattern of change in each variable (time-dose response curves) that may account for the multiple
possible roles of THY in vivo. In the initial phase of the S period, both THY and PUFA
concentrations increased over time. Afterwards, in a second phase, although THY concentration
plateaued both in the egg yolk and droppings, PUFA concentration in the yolk continued
increasing. Likewise, after THY supplementation withdrawal (pS period), while THY
concentration was already depleted, PUFA concentration remain increased. An inverse relation of
patterns was observed for SFA. Thus, these results provide evidence towards the hypothesis
that THY modulation of FA profile is beyond its direct antioxidant activity (i.e. scavenging
free radicals or chelating metal ions; [
]). The further FA-modulation evidenced in the
second phase and during pS can be understood under different rational. First, FA-modulation
can be considered a result of the sum of effects of the direct and indirect THY antioxidant
] in different organs [
]. Second, THY properties could affect other
metabolic pathways that could conduce to PUFA increments [
FA-modulation mediated by the PUFA [
], specifically, in the pS period. In the following paragraphs
these three alternatives are explained in detailed.
In regard to the potential direct antioxidant effect of THY in target tissues (i.e. yolk) it is
important to take into consideration the minimum concentration necessary to provide
meaningful effects. In our study the highest concentrations of THY detected was in the range of 406
ng/g of egg yolk similar to other studies [
], which is equivalent to ~3.05 uM. It has been
14 / 23
Fig 7. Example photomicrographs of quail liver 40X. (A) Normal liver, (B) liver with steatosis or fatty degeneration, (C) vascular congestion and dilatation of sinusoids,
and (D) oval cell focus. Arrows point to representative examples of the corresponding alteration in the photograph.
stated that given a total antioxidant value in human plasma of over 103 mM, a minimum
concentration of 20?50 mM (~36%) of an additional antioxidant from dietary sources would be
required to make a significant contribution to systemic antioxidant capacity . A similar
situation would be found in chickens where antioxidant concentrations in plasma such as
vitamin C and vitamin E would be in a range of 61?66 uM and 13?15 uM, respectively, and total
antioxidant value in plasma could vary from 336.9 mM up to 740?830 mM, considering that
dietary polyphenols result in unconjugated serum levels of up to 1 mM [
]. In the same way,
it has been demonstrated that a concentration of 278 ug of THY/g of yolk was needed to be
added to a control yolk to display an antioxidant activity equivalent to that of a yolk from a
thyme treated hen [
]. Although these authors attributed the increased antioxidant status and
FA modulation observed in vivo to the activity of the other components in thyme, their result
15 / 23
Fig 8. Frequency of histopathological alterations registered in all livers analyzed. The reaction pattern (RP) to which the alterations correspond is
indicated with different intensity of gray and a circular label: RP1 (circulatory disturbances), RP2 (regressive changes), RP3 (progressive changes), RP4
(inflammation) (n = 5; N = 45, only 5 birds from the basal diet were studied).
also could be associated with THY involvement in other mechanisms than the direct
antioxidant activity in the body. It is important to mention that these authors were not able to analyze
the actual concentration of THY in eggs since at that moment they lacked a method to do so.
Considering that no effects of the diet supplied, slaughter batch or their interaction were found, only overall means are shown. Two control (BASAL and VEHICLE) and
three supplemented diets (2, 4, and 6.25 g of thymol/kg of feed). Two slaughter batches were assessed, the first, once finished the supplementation period, and a second
after the post-supplementation period of 21 days. HI = Histopathological index. Liv = Liver. RP = Reaction Pattern; RP1 (circulatory disturbances), RP2 (regressive
changes), RP3 (progressive changes), RP4 (inflammation). The reaction pattern to which the indices correspond is indicated with different grey intensity (n = 5; N = 45,
only 5 birds from the basal diet were studied).
16 / 23
It has been proposed that if polyphenols have direct antioxidant effects in vivo, they might be
capable of exerting such effects more likely within the gastrointestinal tract, where high
concentration of polyphenols may come into direct contact with cells without having undergone
absorption and metabolism [
]. In this context, dietary grape seed proanthocyanidins in rats
] and piglets [
] has been shown to suppress oxidative stress in the intestinal mucosa,
improving general health status. This hypothesis would also be in accordance with the
contention that the chain reaction included in the oxidation of consumed lipids could be inhibited by
the transfer of natural antioxidants such as THY into the female body by feeding, consequently
decreasing the oxidation of constituents transferred into the egg yolk and helping to maintain
]. Thus, THY direct antioxidant activity at gastrointestinal tract level would be
plausible, helping to promote the FA-modulation observed herein. However, the relatively low
concentration of THY detected in target tissues seems to shift the rational from a focus on
direct antioxidant properties to a focus on indirect antioxidant properties, biotransformation,
signaling transduction and gene expression regulation.
The indirect antioxidant effects of THY are based on the potential pro-oxidant activity of
this phenol. In several in vitro experimental systems THY has demonstrated to enhance
reactive oxygen species (ROS) formation, in a dose and time dependent manner [
]. In this
regard, recent studies have shown that administration of moderate doses of epigallocatechin
gallate (EGCG) to mice can produce ROS, which activates the nuclear factor Nrf-2-mediated
induction of antioxidant and other cytoprotective enzymes [
]. Analogous results have
been reported in quail supplemented with EGCG . Whether THY can exert a similar
mechanism in vivo must be proved, but it would be consistent with its known effects on
antioxidant enzymes activity in tissues with high PUFA absolute content (serum, liver, thigh
muscle, brain). For example, in broiler [
] and rats [
], it has been demonstrated that dietary
THY boosted glutathione peroxidase and superoxide dismutase activities along with a decrease
in malondialdehyde levels and an increase in PUFA concentrations, as observed herein.
Similar results have been found in chickens that were fed Cinnamomum cassia [
depending on the scale, pro-oxidant effects can be beneficial and induce FA profile changes.
Other metabolic pathways could also be affected by THY supplementation such as lipids
digestion and fatty acids bioconversion. As it is widely known, birds, like other animals, can
only acquire PUFA precursors (linoleic and alpha-linolenic acids) through the diet, since they
are unable to synthesize them de novo [
]. Once these precursors are in the animal body,
their long chain derivatives (LCPUFA) can be synthesized. Since the supplemented diets were
equal in composition (S3 Table), it could be conceived that THY promotes greater
bioavailability of PUFA precursors through increasing digestive enzyme activities and feed efficiency,
which has been shown to be a dose-dependent phenomena [
]. In addition, THY could
promote greater bioconversion of these precursors in their LCPUFA through increasing
deltadesaturases enzymes activity in the liver. On this subject, it has been demonstrated that
hydroxytyrosol (a natural polyphenol) supplementation in mice enhances liver delta-desaturases,
associated not only with an improvement in n-3 PUFA concentrations but also with a decrease
in oxidative stress biomarkers [
]. Both mechanisms proposed are consistent with our results
where only the two highest doses of THY were able to increase alpha-linolenic acid
concentration, from 14 days of S on, while the three doses induced changes in all long chain-PUFA
(LCPUFA), which began to be observed at 7 days of S.
In relation to the third hypothesis mentioned above, it is important to note that PUFA
regulatory functions have gathered much interest recently [
]. Of great significance to us
are the findings showing that docosahexaenoic and eicopentaenoic acids reduce ROS
generation and increase glutathione, glutamate?cysteine ligase and glutathione peroxidase 4 levels in
astrocytes in a dose-dependent manner (under basal condition and in H2O2-treated) .
17 / 23
Additionally, both n-3 PUFA activated transcription factor Nrf2 also in a dose-dependent
manner. Thus, THY could initially promote increased circulating levels of PUFA but thereafter
also PUFA could be contributing to generate an improved antioxidant status preventing its
own peroxidation. This would explain the changes in FA that continue to occur even when
there is no change in the circulating THY concentration or in the pS period, when the supply
of this compound has been withdrawn. It would be of great importance to investigate the
feasibility of this mechanism after THY supplementation in vivo, since the transcription factor
Nrf2 could become a therapeutic target with beneficial effects in animal physiology [
In our study, since females were fully grown adults in their egg-laying peak, it was expected
that performance traits remained optimal in all treatments [
]. Other previous
studies have reported that mixtures with THY had positive effects on growth performance, egg
production and egg physical characteristics depending on the dose [
], which could be
due to differences in the experimental protocols used, especially regarding the age of the
animals evaluated. Moreover, it has also been suggested that the effects of THY may be greatly
dependent on the diet formulation (affecting digestibility of nutritional components and EOs)
and the environment (challenging conditions such as the firsts weeks of growing, heat stress,
etc.) thus, under optimal conditions, no further performance enhancement could be evidenced
]. Also, we considered positive that THY supplementation did not affect physical egg
traits nor the general well-being and histopathological features of the liver. In this regard, it is
noteworthy to mention that steatosis or fatty degeneration observed in all livers is consistent
with the reproductive status of the females (peak of laying) [
]. We ruled out that it is due
to overfeeding, given daily feed intakes were within the normal range for the species [
and that the composition of the feeds were in accordance with the recommendation for
animals of this age and physiological state [
This is the first study that using pure THY encompasses the in vivo effects of increasing
concentrations of THY during and in the mid-term after chronic supplementation, the
bioavailability of THY in the yolk and droppings, the total FA composition of the yolk and a series of
indicators of productivity and general welfare status in adult quail. With this approach we
showed dose and time dependent effects, that are consistent with THY direct and indirect
antioxidant activities as well as its influence on other metabolic pathways. Thus, supplementation
with THY4 and THY6 promotes the production of eggs with an improved nutritional quality,
whose beneficial effects could be sustained for at least 21 days after a 28-day S period is
finished. Thymol supplementation would be advisable for the production of healthier table eggs,
and fertile eggs. Taken together with previous reports [
], it could also contribute to
successful embryo development. Finally, the doses tested herein do not negatively affect productive
performance traits nor liver histopathology.
S1 Table. Nutrient composition of administered diets.
S2 Table. Vitaminic premix composition of administered diets.
S3 Table. Concentration of feed total fatty acids.
18 / 23
S4 Table. Thymol concentration in supplemented feed and percentage incorporated into
S5 Table. Percentage of thymol transferred to egg yolk and droppings in relation to the
We thank Marcela Palacio and Guillermo Blanco for technical assistance with gas chromatography
analysis and Maria Julia Ortiz and Pablo Prokopiuk for technical assistance with animal husbandry.
Conceptualization: Maria E. Fernandez, Jackelyn M. Kembro, Maria L. Ballesteros, Jorge M.
Caliva, Raul H. Marin, Maria C. Labaque.
Data curation: Maria E. Fernandez, Maria L. Ballesteros.
Formal analysis: Maria E. Fernandez, Maria L. Ballesteros.
Funding acquisition: Jackelyn M. Kembro, Raul H. Marin, Maria C. Labaque.
Investigation: Maria E. Fernandez, Maria L. Ballesteros, Jorge M. Caliva.
Methodology: Maria E. Fernandez, Jackelyn M. Kembro, Maria L. Ballesteros, Jorge M. Caliva,
Raul H. Marin, Maria C. Labaque.
Resources: Jackelyn M. Kembro, Raul H. Marin, Maria C. Labaque.
Supervision: Jackelyn M. Kembro, Maria C. Labaque.
Visualization: Maria E. Fernandez, Jackelyn M. Kembro, Maria L. Ballesteros, Raul H. Marin,
Maria C. Labaque.
Writing ? original draft: Maria E. Fernandez.
Writing ? review & editing: Maria E. Fernandez, Jackelyn M. Kembro, Maria L. Ballesteros,
Jorge M. Caliva, Raul H. Marin, Maria C. Labaque.
19 / 23
20 / 23
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(Eugenol, Carvacrol, Thymol, Borneol, Eucalyptol) of Plants and Intact Rosmarinus officinalis Oil. J
Agric Food Chem. 2014; 62(28):6632?9. https://doi.org/10.1021/jf501006y PMID: 24955655
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