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Veterinary Science Research
https://ojs.bilpublishing.com/index.php/vsr
ARTICLE
Drinking a Structured Water Product on Markers of Hydration, Air-
way Health and Heart Rate Variability in Thoroughbred Racehorses:
a Small-scale, Clinical Field Trial
Michael Ivan Lindinger1* Foster Northrop2
1. The Nutraceutical Alliance, Burlington, ON, Canada
2. Northrop Equine, Louisville, KY, USA
ARTICLE INFO ABSTRACT
Article history
Received: 11 September 2019
Accepted: 14 October 2020
Published Online: 1 December 2020
Racehorses in training are in situations of repeated stress that may have
effects on hydration and health. It was hypothesized that daily consump-
tion of a structured water (SW) product for 4 weeks will result in im-
proved hydration, improved upper airway health and increased heart rate
variability. Two groups of Thoroughbred racehorses matched for phys-
iological, training and racing attributes were studied for 4 weeks. One
group (n = 17) received 10 L (about 15%) of their daily water as SW (fol-
lowed by ad libitum ltered deep well water) and the control group (n =
15) only ltered deep well water. Blood samples and bioelectrical imped-
ance analysis (BIA) measures were obtained at baseline, 2 and 4 weeks.
Hydration was assessed using BIA. The upper airway was assessed by
nasopharyngeal endoscopy at baseline and weekly within 60 minutes of
breezing. On weekly breeze days heart rate was recorded at rest, during
exercise and recovery and data were analysed for heart rate variability.
Compared to controls, horses drinking SW showed increased hydration
improved upper airway health post-breezing and increased heart rate vari-
ability. It is concluded that drinking 10 L daily of SW increased hydration
and may have conferred some wellness benets.
Keywords:
Exercise
Heart rate variability
Bioelectrical impedance analysis
Upper airway
Reactance
Phase angle
Resistance
BIA
*Corresponding Author:
Michael Ivan Lindinger,
The Nutraceutical Alliance, Canada;
Email: mi.lindinger@gmail.com
1. Introduction
As the most important essential nutrient in most
biological systems, water plays central roles in all
facets of cellular, tissue, organ and organism func-
tion. For more than 50 years we have known that water in
biological systems is structured [1-7], and also that structured
water (SW) occurs naturally [8,9] or can be man-made [10,11].
However, we still have very little idea what happens to SW
when it is taken up into plants or consumed by animals.
While the internet is rife with anecdotes, hard science is
lacking. The present study describes the effects of a stable,
man-made SW in an intact biological system, the Thor-
oughbred racehorse in training and competition.
Physicists and chemists have been studying SW for
nearly 8 decades [1-5]. Liquid water can be structured, de-
ned as an increase in the numbers of water clusters, using
variety of methods including magnets, light and other forms
of electromagnetic energy [7,12,13]. Magnetized water (water
treated by being in the inuence of magnets) briey gains
altered water structuring and has received some attention in
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plant and animal agricultural research [14]. While these types
of magnetized waters are stable for only a few hours to a
few days, the studies universally report benefits to plants
and animals unless the intensity of the magnetic eld used
for treating the water is too high [13,14]. It remains unknown
how these types of SW confer their benets.
Some notable studies reporting effects of SW on cell
systems include the following. Culture medium created us-
ing SW, compared to control medium, resulted in increased
viability of mouse splenocytes, increased viability of RAW
264.7 macrophage cells, and doubling of phagocytic ac-
tivity [15]. This study also showed up to a 3-fold increase in
natural killer (NK) cell activity when NK cell activity was
assessed using CCK-8 assay to measure cell cytotoxicity
against YAC-I cells. The authors concluded “these results
strongly suggest that water plays a critical role in cellular
metabolism, more than previously understood.” This view
is elegantly supported in Ball’s [4] perspective on the roles
of SW in living systems. Similar lines of evidence using
cell systems were reported by Lee et al. [16]. Sharma et al. [17]
reported increased rate of seed germination, sapling growth
and development when chickpea seeds were incubated, and
saplings grown, in SW compared to controls.
The present study used multi-frequency bioelectrical
impedance analysis (BIA) for the non-invasive assessment
of hydration and cellular integrity in horses [18-20]. At pres-
ent, the limits of detecting changes in horses are similar to
that in humans: it is not possible to detect changes smaller
than 3% of total body water (TBW) or extracellular u-
id volume (ECFV). Water contents are calculated from
measures of impedance to current flow, and reactance,
over a range of different frequencies. It is likely that direct
measures of reactance (the delay in conduction as a result
of capacitance by cell membranes and tissue interfaces)
which is directly related to membrane capacitance, is able
to provide an index of hydration and cellular integrity at
higher resolution than calculated water volumes [19,20].
Racehorses live in a relatively stressful environment
comprising little daily turnout, periodic withholding of
water, bouts of near-maximal exercise, transport to race-
tracks and being housed in unfamiliar surroundings. In
association with these stressors, many racehorses have
mild to moderate upper airway inflammation which is
associated with poor performance [21] and that often pro-
gressively deteriorates through a horse’s racing career [22].
With efforts to reduce the use of medications by many
racing jurisdictions [23], alternative approaches are being
sought to more naturally support health and performance
of racehorses. The purpose of the present study was to
determine, using a group of actively training and racing
Thoroughbred horses if daily drinking of SW would im-
prove indicators of health including hydration, tissue con-
ductivity, upper airway health and heart rate variability. It
was hypothesized that daily consumption of 10 L of a SW
product increases hydration, improves upper airway health
and increases heart rate variability.
2. Materials and Methods
2.1 Structured water
The Deance structured water - DSW (Deance Brands, Inc.,
Nashville, TN, USA) was made using puried water (reverse
osmosis) to which has been added a very small quantity (less
than 0.01%) of salt (potassium bicarbonate, silica) in order
to stabilize the water structure. This “mineral” water is then
subjected to magnetic and light radiation in a specific se-
quence similar to that described by Lorenzen [11,12].
Aquaphotomics analysis [24] of the SW (referred to as
“magnetized water” in Figure 1) included water boiling (to
determine stability and to determine states of the water at
different temperatures during cooling), infrared spectroscopy
(with spectral analysis), electrical conductivity, dissolved
oxygen and pH. The analysis showed that the DSW had a
unique molecular structure accompanied by increased elec-
trical conductivity, pH and dissolved oxygen, with enhanced
absorbance of specific water molecular conformations
evident from infrared spectroscopic analysis (Figure 1).
Peak points on the spectrogram (Figure 1) indicated proton-
ated water clusters (1379.5nm), trapped water molecules
(1395nm), free water molecules (1411nm), hydrated water
(1417, 1426.5nm) and water dimers (1442.5nm). The sum-
mary from the report stated that DSW was very stable over
time (1 month) even after boiling. The sample when rst test-
ed was already 2.5 months and was re-tested 3.5 months.
Figure 1. Spectrogram of near infrared analysis of the
Deance structured water (indicated by the “magnetized”
red line, and four other water samples of which three are
structured waters (7W, 12W, 16W). The reference (envi-
ronmental control) was MQ (tap water puried by reverse
osmosis). On the red line the wavelengths indicate peaks
of interest: 1379.5nm, protonated water clusters; 1411nm,
free water molecules; 1417 and 1426.5nm hydrated water;
1442.5nm, water dimers
DOI: https://doi.org/10.30564/vsr.v2i2.2380
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2.2 Animals
The study was performed in accordance with the Animal
Protection Laws of the State of Florida (USA) and the
United States Department of Agriculture. Ethical review
was performed by the animal care and use committee of
The Nutraceutical Alliance in accordance with the guide-
lines of the Canadian Council on Animal Care. Horses
were cared for according to standard practices for a Thor-
oughbred race training center. The research was performed
at a training yard in Palm Beach County from mid-Janu-
ary to mid-March 2020. Average day time temperatures
were 22oC with about a 10-15oC diurnal variation. Owner
informed consent was obtained prior to recruitment of
horses into the study.
Horses were individually housed in 4 m X 4 m box
stalls with no ceiling and continuously open door (chained
entry way). Horses were fed about 3kg of timothy hay
twice daily and one to 2 kg of a grain mix twice daily,
once after the morning workout at about 11 am and then
at about 4 pm. Feed was portioned with respect to meeting
the energy and electrolyte needs and maintaining healthy,
race-performance body condition. The diet of each horse
in the study was not changed during the course of the
study and all horses always consumed their entire grain
and hay rations.
The grain was a premium race formula all grain sweet
feed (T913JOC, Jockey, Lexington, KY, USA 40523) that
comprised 13% crude protein (min), 6% crude fat (min),
75% carbohydrate, 12% ADF(max), 21% NDF (max) and
minerals and vitamins. Horses had ad libitum access to l-
tered (activated charcoal) deep (about 150 feet) well water
provided in 15 L buckets. Horses performed a standard
race-training workout in the morning 6 days / week, with
one of these at near race-pace (breezing).
Prior to recruitment into the study all horses received
a veterinary examination that followed the format of the
American Association of Equine Practitioners and that
included endoscopic examination of the upper airway.
Horses were examined in their stall and restrained using
a nose twitch. A one meter long, 10 mm diameter exible
endoscope was passed up the right nostril to the naso-
pharynx and into the trachea to the level of the carina.
The assessments were performed by a single veterinarian.
Observation of locations of redness and swelling were re-
corded, and scoring of tracheal mucous and blood (markers
of upper airway health) were as described by Salz et al.
[21]. At the time of recruitment the following were in the
normal range for resting horses: blood hematology and
biochemistry, heart rate, respiratory rate, rectal tempera-
ture, locomotion, eyes, mucus membranes, g.i. sounds (in-
testinal motility). The characteristics of the horses in each
group are shown in Table 1. There were no significant
differences between the two groups of horses in watering
practices, feeding practices, stall locations with respect
to water supply, handling practices, medications, perfor-
mance, age and gender.
The control group (n = 15, Table 1A) comprised 13
3-year-old horses, 1 aged 4 and 1 aged 6; two were mares
and 13 were geldings. All horses received daily ome-
prazole and sulfacrate (to treat / prevent gastric ulcers),
one received dantrium (to prevent post-workout muscle
cramping), one received Equisul (333 mg sulfadiazine
and 67 mg of trimethoprim / ml; used for treatment of
lower respiratory tract infection); one received Regumate
(Altrenogest 2.20 mg / ml; used to control estrus); one
received Metronidazole (antibiotic). Six horses were rou-
tinely on medication to support airway health, and this
was administered only prior to weekly breezing: furose-
mide (3 to 5 cc) and amicar (1 at 0, 3 at 10cc or 2 at 20
cc).
The DSW group (n = 17, Table 1B) comprised 15
3-year-old horses, 1 aged 4 and 1 aged 6; two were mares
and 15 were geldings. All horses received daily ome-
prazole and sulfacrate, one received dantrium, and one
received an amino acid supplement. Four horses were rou-
tinely on medication to support airway health, which was
administered only prior to weekly breezing: furosemide (3
to 6 cc) and amicar (2 at 10 cc and 2 at 20 cc).
2.3 Research Design
The present descriptive, small-scale, clinical eld trial was
designed to accommodate horses in a race-training situ-
ation and that were available for up to 34 days of contin-
uous use. The design therefore required closely matched
control and treatment group horses similar in all key
physiological, feeding, drinking, training and racing pa-
rameters. A cross-over design could not be accommodated
because of the restricted availability of horses (34 days)
and because the duration of a required “washout” period
was unknown, as it remains unknown how long-lasting
the effects of DSW are. The number of horses required
in each group was determined using a power calculation
with BIA phase angle, resistance and reactance as the key
variables of interest, using data from Ward et al. [20]. The
minimum number of horses required in each group was
18, and because this was a eld study using race horses
in active training and competition it was anticipated there
would be signicant attrition over the 5-week period from
recruitment to study completion.
A total of 40 horses were recruited for the study and as-
signed to two groups so that each group was similar with
DOI: https://doi.org/10.30564/vsr.v2i2.2380
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Table 1A. Characteristics of control group horses recruited into the study at the recruitment stage. Body condition score
of all horses was 3.5
Horse Age Sex H (cm) Medications other than omeprazole and
sulfacrate speed (m/s) Upper airway score Upper airway endoscopy results
34 G 169 17.35 2 slight right side asymmetry, function
normal
43 F 168 equisul 17.75 1
63 F 165 dantrium, furosemide, amicar 17.91 1
83 G 170 furosemide, amicar 17.78 2 grade 3 pharyngitis
12 3 G 164 16.97 1
14 3 G 167 17.24 1
15 3 G 164 furosemide, amicar 17.67 1 accid epiglottis, grade 2 pharyngitis
16 3 G 173 Regumate, furosemide, amicar 17.56 1
18 3 F 167 na 1 slightly accid epiglottis
21 3 F 167 17.76 1
23 3 G 174 furosemide, amicar 17.66 4 left arytenoid paralysed, appears to have
been tied back
25 3 G 167 17.46 1
27 6 G 171 metronidazole 17.98 1
28 3 G 165 17.41 1
29 3 G 168 na 1
30 3 G 170 na 1
mean 168.1 17.58 1.31
SD 2.98 0.28 0.79
Table 1B. Characteristics of Deance structured water group horses recruited into the study at the recruitment stage.
Body condition score of all horses was 3.5
Horse Age Sex H (cm) Medications other than omeprazole and sul-
facrate speed (m/s) Upper airway score Upper airway endoscopy results
13 G 168 furosemide, amicar 17.53 1
26 G 172 omeprazole, sulfacrate, furosemide, amicar 17.84 1
55 G 168 18.32 1
73 G 171 17.31 2 slight right side asymmetry,
good Function
93 F 169 dantrium 17.77 1
10 3 G 172 17.72 1 slightly short epiglottis
11 3 F 163 na 1
13 3 F 165 17.74 1 slightly short epiglottis, grade
2 pharyngitis
17 3 G 170 16.60 1
19 3 G 170 body builder 17.65 1 slightly accid epiglottis
20 3 G 169 16.97 1
22 3 G 169 17.24 1
24 3 G 166 17.46 1
26 3 G 168 16.79 1 thin, slightly short epiglottis
31 3 G 170 na 2 grade 4 pharyngitis
32 3 G 170 furosemide, amicar 17.95 2 short epiglottis
33 3 Male na 1
34 3 Male 164 16.98 1
35 4 Male 169 17.35 2 slight right side asymmetry,
function normal
36 3 Female 165 dantrium, furosemide, amicar 17.91 1
37 3 Male 170 furosemide, amicar 17.79 2 grade 3 pharyngitis
38 3 Male 165 17.42 1
39 3 Female 168 equisul 17.75 1
40 3 Female 167 na 1 slightly accid epiglottis
mean 168.2 17.51 1.21
SD 2.47 0.43 0.41
Notes: H = height; G = gelding; F = female (mare); na = not available
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respect to all parameters (as noted above) at the start of
the study. The control group was assigned 16 horses and
the DSW group 24 horses. One horse was lost from the
control group and 7 horses were lost from the DSW group.
Reasons included: horse moved off-property (6), illness
(1), lameness (1). Because attrition was not balanced with
respect to characteristics, the two groups varied slightly,
as described below.
2.4 Study Execution
Following recruitment into the study the horses were
housed in one shed row with large eaves that provided
shading while permitting ample air movement. The study
comprised a baseline period of 4 days, followed by 28
days of receiving placebo or DSW. On two separate days
during the baseline period blood samples were taken from
the jugular vein and bioelectrical impedance readings
were taken.
During the 28 days of the trial, the horses in the DSW
group received 10 L of DSW in their normal water bucket
as the rst water they consumed after the morning work-
out. This was followed by consumption of normal ltered
groundwater ad libitum throughout the remainder of the
day. Horses in the control group drank only the filtered
deep well water, ad libitum. It was not practical nor possi-
ble to measure individual water intakes, nor was this nec-
essary with the experimental design. Water intake is only
one part of the uid balance equation, which also includes
significant losses of water through the respiratory tract,
skin and kidneys in athletic horses.
During the baseline period, and throughout the fol-
lowing 28 days, when a horse was to perform a breezing
workout (weekly near race-pace gallop), it was rst tted
with a heart rate monitor (Polar H10 with the equine strap;
(Polar Electro Oy, Kempele, Finland). The H10 monitor
was connected via Bluetooth wireless to the Polar Equine
application (Polar Electro Oy, Kempele, Finland) using
android smartphones. This enabled the collection of heart
rate data to the smartphone while the horse was resting,
warming up, galloping and cooling down. Breezing was
performed by all horses typically weekly, but not all hors-
es performed a breezing workout each week. All horses
performed a daily submaximal workout, with one rest day
per week.
Some horses from each group (n = 7 DSW; n = 6 con-
trol) went off-site for 1 to 4 days to compete in races;
individual horses did not compete in more than one race
during the 28 days. When horses went off-site they drank
the local water, and DSW group horses did not receive the
DSW product during this time.
2.5 Blood Analyses
Blood hematology and biochemistry was performed in or-
der to determine if 28-day consumption of DSW affected
any clinical parameters associated with these blood vari-
ables. Blood samples were collected into 3 cc vacutainer
tubes containing EDTA for CBC / hematology (CELL-
DYN 3700, Abbot Diagnostics, Lake Forest, Ill, 60045
USA) or without additive for analysis of serum biochem-
istry (Envoy 500, ELITech Group Inc., Smithfield, RI
02917 USA) at On Track Lab Inc. (Lake Worth, FL 33449
USA) within 2 hours of sampling.
2.6 Bioelectrical Impedance Analysis (BIA)
Bioelectrical impedance measures were obtained on two
separate days mid-way through the study (days 13 to 15,
between 3:00 and 4:30 pm) when the horses were resting,
post-prandial in their stalls prior to the afternoon feeding.
All BIA measures were performed by the same individu-
als to ensure consistency of electrode placement and very
calm handling of the horse. The people performing the
BIA measures were blinded to the treatment. Horses were
not restrained prior to and during measurements. At the
end of the study blood samples were obtained about 30
minutes prior to BIA, followed by BIA measurement on
each of days 28 and 29. Heart rate data continued to be
collected until day 31.
Horse height was measured to the top of the withers
using an equine height measuring stick as described pre-
viously [25,26]. Body condition score was the same in all
horses, being a 4 on the 9-point scale [27] - these were lean,
well-muscled, very t horses. Bioelectrical impedance anal-
ysis was performed using an Equistat 5000 multifrequency
bioelectrical impedance analyzer (Bodystat Ltd, Douglas,
Isle of Man, IM4 4QJ, British Isles) as described previously
[25,26]. The hair coat on all horses was very short due to the
climate, and very clean. Two 10 cm2 carbon bre electrode
pairs (10 cm between centers) were dabbed with an ade-
quate amount of conductive gel to penetrate to the skin,
then were placed above the left fore- and hindlimb above
the knee and hock, respectively, overlying the prominent
muscle. On the forelimb, the electrode pair was positioned
proximal to the carpal joint on the lateral portion of the ra-
dius directly over the common digital extensor, ulnaris lat-
eralis and radial carpal extensor muscles. On the hindlimb,
the electrode pair was placed on the tibia directly over the
long digital extensor and lateral digital extensor muscles.
The electrodes were held in place using a cuff secured by
Velcro straps. Shielded leads connected the electrodes to
the BIA instrument. BIA was performed using a tetra-polar
arrangement in which an 800 µA alternating current was
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applied through the body using the 2 distal electrodes, and
voltage drop measured by the 2 proximal electrodes. The
instrument recorded impedance (Z), resistance (R), reac-
tance (Xc) and calculated phase angle (PA) at 7 frequen-
cies: 5, 16, 24, 50, 140, 200 and 280 kHz [26].
Measurements were repeated two to six times (depend-
ing on whether the horse moved during measurement),
without removing the electrodes, until similar impedance
values were obtained at least twice in sequence. Occasion-
ally, when a horse moved a limb, one or more electrodes
had to be adjusted in order to re-establish good contact
between skin and electrodes and obtain a repeatable
measurement series. The entire procedure for two scans
required less than 2 minutes. The instrument’s algorithms
were developed using primarily Standardbred and Thor-
oughbred horses, and were used to calculate body mass,
TBW, ECFV and ICFV [26]. The average values of two
readings acquired when horses were standing quiet and
still were used for subsequent data analysis.
The raw impedance parameters R and Xc were used
to corroborate the algorithm-computed hydration param-
eters; this is relevant because R and Xc were not used in
these algorithms [26]. The bioelectrical impedance vector
analysis (BIVA) approach developed by Piccoli et al. [27]
has been used extensively in assessment of acute changes
of hydration [27,28,29].
2.7 Post-breezing Nasopharyngeal Endoscopy
On days when horses were breezed, they received a na-
sopharyngeal endoscopy within 60 minutes of finishing
breezing. One veterinarian performed the procedure on
all the horses during the study duration. The veterinarian
was blinded with respect to the treatment and assessed
the upper airway. Observations were graded as described
by Salz et al. [21]. The amount of mucus in the trachea was
graded using a score of 0-4 (0, no mucus; 1, small, singu-
lar threads or droplets of mucus; 2, larger, conuent drop-
lets of mucus. The amount of blood in the trachea was
similarly graded using a score of 0 to 4 (0, no blood de-
tected in the pharynx, larynx, trachea or main-stem bron-
chi; 1, presence of one or more ecks of blood or two or
less short, narrow streaks of blood in the trachea or main-
stem bronchi). No horses had mucus greater than grade 2
or blood greater than grade 1. The upper airway was also
observed at the same time for presence of chondritis, dor-
sal displacement of soft palate, and inammation (swelling
and redness) of the left arytenoid.
2.8 Heart Rate Variability (HRV)
Data for each horse for each day that they were breezed
were organized by treatment (Control, DSW), study dura-
tion (0, 1, 2, 3, 4 weeks) and activity (rest, canter, full gal-
lop, recovery). Raw txt les downloaded from the Polar
Equine app were imported into an HRV analysis program
(Kubios HRV Standard 3.3.1, Kupio, Finland) which l-
tered the data to remove artifacts. Approaches similar to
that described previously [29-31] were used to select the time
periods for HRV. Data for analysis of HRV were selected
from the entire heart rate trace during a 3-minute period at
rest, for a 3-minute period when the horse was in the can-
tering phase of the warm-up, for 30 s when at full gallop,
and for a 5 minute period in recovery.
2.9 Statistics
The person that performed the data entry and statistics
were blinded to the treatment group. The statistics pro-
gram (SigmaPlot 14, Systat Software Inc.) tested the data
for normality and kurtosis (Shapiro-Wilk test). Treatment
and time effects for the endoscopy data, BIA data, and
blood data were assessed using two-way analysis of vari-
ance. Within-treatment effects were examined using one-
way, repeated measured analysis of variance. For the HRV
data, complete data sets were obtained from only three
horses in each group in all of the three categories of treat-
ment, week and activity. Missing data were the result of
inadequate signal detection, illness, off site to race, and
attrition from the study. Thus 2-way RM-ANOVA with
respect to treatment and study duration was not possible.
The entire data set was used for the analysis in order to
provide adequate data for each week and for each activity
level. Comparisons were assessed using a 2- way ANOVA
(treatment, week). Activity was a confound because of the
large variation from rest to full gallop. For effects within
each activity level a 2-way ANOVA was performed with
respect to treatment and week. When a signicant F-ratio
resulted, then a 1-way RM-ANOVA (horse as subject) was
performed within treatment and activity. The Bonferoni
post-hoc test was used for because of its ability to handle
missing data. Data are presented as mean + SD. Signi-
cance is at p < 0.05 at a power of 0.8 or greater.
3. Results
3.1 Upper Airway
The results of the upper airway endoscopic examination
showed that in the control group there was no change
over time. There was a signicant difference between the
control and DSW groups. In the DSW group there was an
increase in the number and percentage of horses that had
an improved upper airway result (Table 2, Figure 2).
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Table 2. Results of nasopharyngeal endoscopy performed
30 - 45 minutes after breezing. Results are only from
horses that completed the entire study
DSW Horses
(n = 17)
Week 0 Week 1 Week 2 Week 3 Week 4
Mucus - 1 4 5 2 2 3
Mucus - 2 2 1 3 0 1
blood - trace 4 1 2 0 1
blood 1 1 0 0 0 0
SLC 2 0 2 1 1
chondritis 0 1 1 0 0
ddsp 0 0 1 0 0
OK 7 5 7 11 10
n = 16 14 16 14 14
% with clear
airway 43.8 35.7 43.8 78.6 71.4
Control Horses (n = 15)
Week 0 Week 1 Week 2 Week 3 Week 4
Mucus - 1 3 3 3 4 3
Mucus - 2 1 0 1 0 0
blood - trace 1 1 3 2 4
SLC 1 3 0 2 0
chondritis 0 0 0 0 0
ddsp 0 0 0 0 0
OK 7 7 7 7 2
n = 12 12 12 13 8
% with clear
airway 58.3 58.3 58.3 53.8 25.0
Notes: ddsp = dorsal displacement of soft palate
SLC = slight inammation of the left arytenoid (slight left cord)
DSW - Deance structured water
OK indicates the absence of adverse observations (clear airway).
Time (weeks)
0123 4
% of horses with no ob servable airway c oncerns
20
30
40
50
60
70
80
90
SW
Control
*
*
Figure 2. Percentage of horses that had no observable
indications of impaired airway health (see Table 2) upon
examination after breezing in the control (n = 15) and
DSW (n = 17) groups. * indicates signicantly greater
than in the control group and signicantly greater that at
week 2 in the structured water (SW) group
3.2 Performance Indices
Training speeds did not change over time and there was
no significant difference between groups (Control 12.4
+ 0.3 seconds / furlong; DSW 12.4 + 0.3 s/ f). Breezing
distances ranged from 3 to 6 furlongs. For horses from
each group that competed in a race, speeds were also not
signicantly different (Control 11.9 + 0.2 s / f; DSW 11.9
+ 0.5 s / f) over distances ranging from 6 to 8.5 furlongs.
Note that horses only raced once in a 4-week period and
races occurred during each of these 4 weeks so it is not
possible to determine a treatment effect.
3.3 BIA - Hydration Parameters
In the control group TBW increased from 333 + 6 L to 348
+ 6 L at 2 weeks, then decreased 342 + 19 L at 4 weeks
(Figure 3). In the DSW group TBW continued to increase
from baseline (336 + 19 L) to 2 weeks (353 + 19 L) to 4
weeks (360 + 6 L); the increase in TBW in the DSW group
was significantly greater than in controls. At 2 weeks, in
both groups, the increase in TBW was primarily due to in-
creased ICFV, as ECFV did not increase signicantly (Fig-
ure 3). In the control group the decrease in TBW from week
2 to week 4 was mirrored by the decrease in ICFV, but
ECFV was increased at 4 weeks. In the DSW group there
was no change in ECFV, while ICFV continued to increase
from 2 to 4 weeks. Baseline values for ECFV were 124 + 5
and 124 + 6 in the control and DSW groups, respectively.
Baseline values for ICFV were 211 + 16 and 216 + 14 in
the control and DSW groups, respectively.
Figure 3. Changes in BIA-predicted whole body hydra-
tion parameters from baseline (0) to 2 and 4 weeks in
total body water (TBW, circles) intracellular uid volume
(ICFV, squares; error bars omitted for clarity) and extra-
cellular uid volume (ECFV, diamonds) in the control
group (black, n = 15) and the DSW group (blue, n = 17).
Values are mean + SD. DSW = Deance structured water;
* indicates signicant increase from baseline. ** indicates
signicant increase from baseline and compared to the
opposing group
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Plasma volume (PV) was similar at baseline in the con-
trol (18.7 + 1.3 L) and DSW (18.9 + 1.1 L) groups. PV
increased signicantly more in DSW at 2 weeks (20.2 + 1.1
L) and 4 weeks (20.6 + 1.2 L) than in controls at 2 weeks
(19.4 + 1.3 L) and 4 weeks (19.0 + 1.4 L). In the DSW
group the increase at 4 weeks represented a 9.2% increase
in PV, contributing to increased circulating blood volume.
In controls, the increase in uid volumes were accom-
panied by increases in calculated body mass as follows
from 466 + 14 kg (baseline) to 490 + 14 (p < 0.05) and
487 + 15 kg at 2 and 4 weeks, respectively. In the DSW
group, calculated body mass increased from 479 + 12 kg
(baseline) to 505 + 12 (p < 0.01) and 513 + 13 kg at 2 and
4 weeks, respectively. At 4 weeks the increase in calculat-
ed body mass in the DSW group was greater than in con-
trols (p < 0.01 ). At each time point the increase in TBW
was 65 to 70% of the increase in calculated body mass.
3.4 BIA - Bioelectrical Parameters
The mean impedance-frequency relationships (IFR) for
both groups at baseline, 2 and 4 weeks are shown in Figure
4. At baseline there was no signicant difference between
groups in the IFR. At 2 weeks in the control group there
was a modest and signicant decrease in impedance at each
frequency, but no significant difference between baseline
and 4 weeks and no signicant difference between 2 and 4
weeks. In the DSW group there was a signicant decrease
in impedance at each frequency from baseline to 2 weeks,
and a further signicant decrease to 4 weeks, with no sig-
nicant difference between 2 and 4 weeks.
Resistance (R) and reactance (Xc) were measured and
phase angle (PA) calculated by the instrument at only the
50 kHz frequency. At this frequency relatively little current
passes through cells. Resistance is the decrease in voltage
reecting conductivity through ionic solutions. Reactance
is the delay in the ow of current measured as a phase-shift,
reecting dielectric properties mainly attributed to capaci-
tance of cell membranes and tissue interfaces [20,29].
The signicant difference between groups and the time
course of change in the IFR led to an analysis of the time
course of change in R, Xc and PA. Hydration parameters
can change in the absence of change in Xc and PA, and this
remained to be determined. Figure 5 shows the time course
of change in R and Xc in control and DSW horses. In con-
trols, there was a small signicant decrease in R at 2 weeks
but not at 4 weeks, and there was no change in Xc or PA (not
shown). In the DSW group, there were signicant decreases
in both R and Xc at 2 and 4 weeks, with no change in PA.
The PA of horses was not significantly different between
groups nor at any time point; pooled data are described as
14.7 + 0.6 ( range 13.1 to 16.5; median 14.7).
Input frequency (kHz)
050 100 150 200 250 300
Impedance (Ohms)
60
70
80
90
100
110
120
Control baseline
Control mid
Group vs Col 7
Control end
SW mid
Group vs Col 7
SW end
Figure 4. Impedance-frequency relations in DSW and
control groups horses at baseline, mid- study (2 weeks) and
end-study (4 weeks). There was a signicant decrease in
impedance at each frequency in DSW horses at mid-study (2
weeks) that was maintained at end-study (4 weeks)
Figure 5. Time course of whole body resistance (top pan-
el) and reactance (bottom panel) measured at a frequency
of 50 kHz. DSW = Deance structured water; * indicates
signicantly different than baseline; ** indicates signi-
cant difference between groups
Analysis of the combined data sets for control and
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DSW horses yielded significant linear relationships be-
tween predicted uid volume parameters (only shown for
TBW, Figure 6) with respect to R and Xc (Figure 6 top
and middle panels), and a significant linear relationship
between Xc and R (Figure 6, bottom panel). When exam-
ined this way, there was no signicant difference between
groups, and the data are used to illustrate the relationships
between bioelectrical and physiological parameters.
Figure 6. Top panel: the relationship between resistance
(R) and total body water (TBW): R = 179.5 - TBW x
0.261; r2 = 0.787; p < 0.001. Middle panel: the relation-
ship between reactance (Xc) and TBW: Xc = 13.92 -
TBW x 0.0191; r2 = 0.524; P < 0.001. Bottom panel: the
relationship between reactance (Xc) and resistance (R) at
the frequency of 50 kHz. Data from all horses at all time
points. Xc = 0.490 + R x 0.0763; r2 = 0.721; P < 0.001
Figure 7. Top panel: RXc mean (+ SD) graph for horses
in the DSW group (n = 17). The dashed line indicates di-
rectional change of hydration without loss or gain of soft
tissue mass. The solid arrow indicates the vector direction.
Bottom panel: RXc graph of individual DSW horses at
baseline (black squares) and at 4 weeks (green circles). R
/ H, height-adjusted resistance; Xc / H, height- adjusted
reactance
In the DSW group, there was a simultaneous decrease
in R and Xc without change in PA. In order to better
understand this the BIVA mean graph (Figure 7, top
panel) shows the vector of change in R and Xc directed
downwards and to the left, thus indicating primarily an
increase in hydration with perhaps some increase in soft
tissue mass. Cluster presentation of individual data clearly
demonstrates the downwards shift to the left (Figure 7,
bottom panel).
3.5 Heart rate variability (HRV)
The only signicant differences between treatment groups
in HRV occurred in horses standing quietly at rest in the
stall. Resting heart rate did not change over time in the
control group. At baseline, week 1 and week 2 there was
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no signicant difference between control and DSW groups
(Figure 8). At weeks 3 and 4 resting heart rate in the DSW
was signicantly lower than at baseline, and signicantly
lower than in the control group.
Figure 8. Resting heart rate in horses receiving 10 L / day
of DSW (n = 7 to 18) and controls (n = 6 to 11). DSW =
Deance structured water; * signicantly different from
baseline within treatment; ** signicant different between
treatments
There was no significant difference between groups
in the beat to beat (RR) interval due to the rather large
variability in the control group (Figure 9). However, in
the DSW group there was an increase in RR interval over
time, so that at 3 and 4 weeks it was signicantly elevated
compared to baseline. The progressive lowering of rest-
ing heart rate and lengthening of RR in DSW horses was
associated with an increase in the PNS Index (Figure 10)
and a decrease in the SNS Index (Figure 11).
Figure 9. The beat to beat (RR) interval in horses receiv-
ing 10 L / day of Deance structured water (DSW; n = 7
to 18) and controls (n = 6 to 11). * signicantly different
from baseline within treatment
Figure 10. The index of parasympathetic nervous system
activity (PNS Index) to cardiac control in horses stand-
ing at rest. DSW horses received 10 L / day of Deance
structured water (DSW; n = 7 to 18) and controls received
normal water (n = 6 to 11). * signicantly different from
baseline within treatment
Figure 11. The index of sympathetic nervous system
activity (SNS Index) to cardiac control in horses stand-
ing at rest. DSW horses received 10 L / day of Deance
structured water (DSW; n = 7 to 18) and controls received
normal water (n = 6 to 11). * signicantly different from
week 1 within treatment
3.6 Blood biochemistry and hematology
There was no signicant difference between baseline and
end-study for both treatment groups, and no significant
differences between treatment groups (Table 3).
An analysis was performed using all horses that had
elevated concentrations of muscle enzymes, although the
concentrations were not markedly elevated and in the ex-
pected range for regularly exercised Thoroughbred horses
in training. This analysis was performed to determine if
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daily consumption of DSW would reduce the appearance
of muscle enzymes. All horses that had an AST of 400
IU/L and higher were selected for analysis, and correla-
tions analyzed with respect to days since breezing and
concentrations of each of the 4 enzymes. There was no re-
lationship between days since breezing and enzyme con-
centration. There were no signicant differences between
baseline and end-study for muscle enzymes in the two
treatment groups.
4. Discussion
The present study showed that horses that drank DSW
displayed improved hydration by two weeks, with signi-
cant increases in TBW, ECFV, ICFV and plasma volume.
In 50% of the DSW group horses having signs of upper
airway health concerns at the start of the study, there
was an improvement (reduction of number and severity
of marker) of airway health. There were no effects on of
performance, blood biochemistry and hematology, not
any adverse events. By 3 weeks, the DSW group horses
showed an altered autonomic response characterized by
lower heart rate, increased RR interval, increased PNS
index and reduced SNS Index. The present study indicates
that it is not necessary to drink all water as “beneficial”
Table 3. Blood biochemistry and hematology in horses in the control group (n = 15) and Deance structured water (DSW)
group (n = 17). Standard deviation = SD
Control
Baseline
Control
End
DSW
Baseline
DSW
End
Mean SD Mean SD Mean SD Mean SD
Glucose mg / dl 116.25 8.08 116.45 7.98 112.73 8.60 113.85 8.29
BUN mg / dl 15.36 1.70 14.76 1.68 15.73 2.10 14.74 2.30
CRE mg / dl 1.56 0.18 1.51 0.18 1.55 0.16 1.48 0.19
BUN/CRE ratio 9.92 1.51 9.85 1.15 10.23 1.42 10.02 1.26
Na mmol/L 137.9 3.4 137.8 2.9 137.6 3.3 137.1 2.9
Kmmol/L 3.87 0.24 3.75 0.28 3.72 0.30 3.79 0.29
Cl mmol/L 99.14 2.85 98.76 3.03 98.82 2.77 99.65 2.39
TCO2 mmol/L 29.07 1.22 29.17 1.26 29.07 1.07 29.15 0.99
CA mg / dl 12.11 0.58 11.87 0.54 12.13 0.58 12.07 0.62
Pmg / dl 4.02 0.32 3.92 0.33 4.02 0.30 3.99 0.31
TP g / dl 6.18 0.31 6.01 0.29 6.13 0.35 5.90 0.76
ALB g / dl 4.05 0.16 3.93 0.17 3.95 0.16 3.90 0.41
GLB g / dl 2.13 0.21 2.08 0.23 2.18 0.27 2.23 0.40
A/G ratio 1.92 0.20 1.91 0.24 1.83 0.23 1.80 0.25
BILIRUB mg / dl 2.50 0.73 2.60 0.68 2.53 0.70 2.65 0.77
ALK/PHOS IU / L 158.43 15.43 155.38 23.99 153.09 28.10 151.91 21.70
CK IU / L 340.57 433.47 359.03 431.05 256.77 90.18 335.62 353.82
AST IU / L 486.14 387.94 458.93 320.59 424.00 280.31 504.88 423.13
GGT IU / L 35.86 13.05 36.66 13.15 37.41 9.25 41.18 15.47
WBC x10^3 / ul 8.53 1.46 8.48 1.37 8.63 1.58 8.57 1.54
RBC x10^6 / ul 9.51 0.66 9.55 0.68 9.57 0.58 9.55 0.62
HGB g / dl 14.36 1.02 14.40 1.00 14.51 0.83 14.48 0.91
HCT % 41.3 3.0 41.4 3.1 41.7 2.5 41.6 2.8
MCV fL 43.2 0.92 43.3 0.79 43.19 0.88 43.45 0.64
RDW % 25.7 1.1 25.7 1.1 25.9 0.9 25.8 1.2
PLT x10^3 / ul 153.6 26.5 163.0 36.4 175.8 35.4 172.2 38.9
SEG.NEU % 55.3 8.9 56.3 6.2 55.3 8.6 55.8 10.7
LYM % 43.2 9.0 42.4 6.2 42.9 8.4 42.4 10.4
EOS % 0.9 0.7 0.8 0.7 1.0 1.1 1.0 1.2
MONO % 0.5 0.6 0.4 0.5 0.8 0.7 0.7 0.6
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SW, so long as an adequate amount is ingested, however
high-quality dose-response studies have yet to be per-
formed.
Limitations of the present study are related to the fact
that this was a eld trial that permitted minimal researcher
invasiveness to routine racehorse practices at this training
facility. The purpose of the control group was therefore
to control for those independent variables that could not
be controlled, notably the inability to measure 24/7 wa-
ter intake and losses (respiratory, skin, renal) and body
mass using a weigh beam. Despite the confounds of some
horses going off-site to compete, the duration off-site, the
inability to consume DSW when off-site and the loss of
horses from study groups, the results demonstrated that
DSW has physiological effects which may be deemed as
beneficial in racehorses. These limitations may also be
taken as a positive feature in that the study was performed
in actual eld conditions and the results are thus readily
applicable to horses in active training programs.
Chronic stress, such as that experienced by racehorses
in training and competition, may result in chronic inam-
mation in many tissues and impair cell health and perfor-
mance. An unexpected finding of the present study was
the reduction of signs commonly associated with mild-
to moderate upper airway inammation in racehorses [21-
23]. None of the horses in the study had more than mild
upper airway symptoms, however 50% of the horses that
consumed DSW that initially had upper airway symptoms
showed an absence of symptoms at 3- and 4 weeks. This
may be of practical signicance because airway health has
been reported to progressively worsen during the racing
career of horses [22], and the presence of airway symptoms
is causally related to impaired racing performance [21].
It is generally recognized by the scientic community,
although not generally recognized by horsemen involved
in training, that competitive horses in training undergo
periods of what may be considered as mild dehydration
[25]. There are many reasons for this, the main ones in-
clude withholding of water prior to morning workouts, no
provision of water during transport to competition, and
withholding of water at competition. Again, in science,
this is counterintuitive because it is known that health and
performance are correlated with hydration [33], although
this has not been systematically studied in horses. The
standard operating premise is that (race) horses are able
to tolerate some level of dehydration (up to 5%) without
adverse effects, and that the associated loss of body water
(mass) enables the horse to run faster or sustain speed for
a longer time. However this aspect of equine physiology
requires systematic study.
Using BIA, the present study demonstrated in DSW
horses signicant increases in hydration variables by two
weeks that were sustained through weeks 3 and 4. Based
on the timing of BIA measurements (at 2- week intervals)
it remains unknown how fast the hydration response oc-
curs, and it is possible that a higher level of hydration
may have occurred. Rehydration of exercise-dehydrated
Standardbred horses showed that hydration at 18 hours
increased above that seen at baseline [25], indicating the
potential to further hydrate horses that were not seemingly
dehydrated at the start of the experiment. This is similar
to the present study, where at baseline there were no signs
of dehydration, including no elevation of plasma protein
concentration, and no clinical signs of dehydration in
control group horses at the end of the study. The increased
apparent hydration in DSW horses at 2 and 4 weeks ul-
timately has to be due to increased water retention in
the body, as increased intake could easily be matched
by increased urine output. Necessary assumptions for
the correct estimation of body hydration (composition)
include homogenous composition, fixed cross- sectional
area and consistent distribution of current density [34]. In
these healthy horses of both groups it is likely that these
assumptions were achieved, and that the signicant differ-
ences in hydration parameters from weeks 2 to 4 do repre-
sent an enhanced hydration with consumption of DSW.
The raw impedance parameters R and Xc were used in
the BIVA approach to assess acute changes of hydration.
In the present study, the integrity of R and Xc as indepen-
dent indicators of hydration are supported by the fact that
there were no significant differences between groups in
the relationship between either the R vs TBW, the Xc vs
TBW, nor for any of the other hydration variables ECFV,
ICFV and PV (data not shown). The BIVA plot for horses
in the DSW group (Figure 7) clearly shows the signicant
increase in hydration that occurred, and there was no ef-
fect in control horses.
It should be expected that the PA did not change in
response to consumption of DSW. In healthy humans, the
main determinants of PA are age, sex and body mass in-
dex [34], and these parameters were very similar in the two
groups of the present study. PA is calculated as its arc tan-
gent, (Xc/R) 180o/π, and represents both the amount and
quality of soft tissue. As such, it is had been validated and
used as an indicator of cellular health. Higher values re-
ect higher cellularity, cell membrane integrity and better
cell function. In healthy humans the PA ranges between 5
and 7 and values as high as 9.5 have been reported in hu-
man athletes [35]. Mean (+ SD) of the PA of athletic horses
in the present study was 14.7 + 0.6 (range 13.1 to 16.5;
median 14.7), substantially greater than in humans but
less than obtained in individual muscles of the horse (range
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from 10 to 25 [19]).
Analysis of heart rate variability (HRV) provides in-
formation on the magnitudes of parasympathetic and
sympathetic neural drive affecting autonomic control of
physiological systems, including heart rate [32]. The only
signicant differences between treatment groups occurred
in DSW group horses standing quietly at rest in the stall.
It is likely that the intensity and variability of exercise
would have overwhelmed differences present at rest.
The combination of decreased heart rate, increased RR
interval, increased PNS Index and decreased SNS Index
indicate effects on the autonomic control of the cardiovas-
cular system [36,37]. The autonomic nervous system (ANS)
consists of two branches, the parasympathetic and sym-
pathetic, that together controls the function of internal or-
gans (e.g., heart rate, respiration, digestion) and responds
to disturbances. The inuence of the parasympathetic ner-
vous system is represented by the PNS Index, while the
sympathetic nervous system is represented by the SNS In-
dex in HRV analysis. The parasympathetic nervous system
(PNS) is the “rest and restorative” system that maintains a
low resting heart rate and restorative state when sleeping
or relaxing. The sympathetic nervous system (SNS) is the
“ght or ight” system that responds to large disturbances
by acceleration of heart rate. The present results suggest
that horses regularly drinking DSW developed a slower
and more restful or restorative cardiovascular response
than control horses, and this effect was manifest at 3 and 4
weeks of consuming DSW.
Perspective
The mechanisms by which daily drinking of SW affects
hydration and health are not understood, and the present
study was not designed to address mechanism. What is
known, is that how water interacts with solutes, colloids
and “fixed” proteins determines the attributes and func-
tions of all of the uids, cells and tissues in the body. The
how is influenced by the “water kind”, a term used by
Lenormand et al. [38] to acknowledge different structural
states. Water is not simply bulk fluid within the body -
rather water molecules form coherent clusters and layered
sheets [4,6,7] around molecules and in association with both
polar and non-polar moieties [3,39]. At present, it remains
unknown how ingested SW distributes within the body, or
if there is a preferential retention of SW. The polar, protic,
and amphoteric attributes of water contribute to complex
structural and dynamic characteristics that allow water to
interact in so many ways with biomolecules [4,5]. Several
models for structured or clustered water have been pro-
posed for biological systems - we know that they are pres-
ent and important [2,7], although we have yet to elucidate
much about molecular-level structures and interactions [39].
We do know that these coherent associations of structured
water with other molecules directly affect things like pro-
tein conformation and therefore protein function [40,41], and
ultimately multimolecular networks such as membranes,
signal transduction pathways and biochemical pathways
[41,42]. This includes structural proteins that form cytoskel-
etal networks [38] as well as enzymes that are involved in
the regulation of biochemical reactions. For example, the
dynamics of the living cytoskeleton is “directly” linked to
the “dynamics of water” such that cytoskeletal “networks
are slaved in a direct fashion to fluctuations arising in
intracellular water” with acknowledgment that changing
“water kind” impacts living cells [38]. Angel et al. [43] pro-
posed “that ordered waters contribute to the functional
plasticity needed to transmit activation signals” from the
cytoplasmic face of rhodopsin. This can be extended to
observations describing interactions of water with the
cytoskeleton during the process of cell volume regulation
[44] although they did not explore the inuence of “water
kind”. The unique results of the present study provide
evidence, albeit indirect at this point, that ingestion of SW
alters how water behaves in the body and suggests that
ingested SW retains some physicochemical properties that
affect interactions of this water with molecules including
endogenous water kinds. Future studies are aimed at eluci-
dating main effects using cellular physiology techniques.
The horses in the control group were clinically euhy-
drated, yet ingestion of DSW increased hydration in the
DSW group horses without adverse effects and arguably
with benecial effects. How does hydration improve when
it is deemed to be already adequate? It is known that SW,
whether as small clusters or larger sheets has a greater
density than bulk phase water, and these denser water
structures have a greater ability to interact with mole-
cules, and hence membranes, in biological systems. We
propose that ingestion of DSW results in a re-structuring
of water within the body, and the manifestation of this is
an increased hydration with attendant effects on cells and
tissues that presented as healthier airways and increased
HRV in the present study.
5. Conclusions
When Thoroughbred racehorses drank 10 L / day of DSW,
compared to control horses, they became more hydrated,
had improved airway health, and a greater HRV when at
rest. There were no effects on performance measures, no
effects on blood hematology and biochemistry, nor any
adverse events. The daily consumption of DSW conferred
physiological and health benefits in competitive Thor-
oughbred racehorses.
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Author Contributions
The research was designed by MIL and reviewed by FN.
Veterinary examinations for recruitment were performed
by FN. MIL analysed the data, FN assisted with the inter-
pretation. MIL wrote the manuscript, FN assisted with and
approved revisions.
Funding
This research was funded by Deance Brands Inc, Nash-
ville, TN, USA.
Acknowledgments
The authors are grateful for the assistance provide by staff
at the training centre, especially IJ, LV and AH. We also
thank Dr. Wendy Pearson and Dan Maddox for helpful
discussions and comments on the manuscript.
Conicts of Interest
The Nutraceutical Alliance Inc., to which MIL is an employ-
ee, was contracted by Deance Brands Inc. (Nashville, TN,
USA) to design and execute the study. MIL does not benet
outside of this contractual work. FN declares no conicts of
interest. MIL and FN declare no other competing interests.
The funders had no role in the design of the study; in the col-
lection, analyses, or interpretation of data; in the writing of
the manuscript, or in the decision to publish the results.
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