Exogenous Phytase Plus Cellulase and Phosphorus Excretion in Lactating Dairy Cows
ABSTRACT The objectives were to assess the ef-fects of exogenous phytase plus cellulase on P excretion in lactating cows. The ef-fects of an exogenous phytase plus cellu-lase mixture and dietary P content on P partitioning and excretion were evaluated in nine early lactation cows (mean = 27 d in milk); six of the cows were rumi-nally cannulated. Cows were assigned to treatments in replicated (three) 3 × 3 Latin squares, and each cow received each treatment sequentially in three, 21-d periods. Diets were 45% forage (all corn silage) and included supplemental P (high P; 0.47%), no supplemental P (low P; 0.32%), or no supplemental P with ex-ogenous phytase (low P-enzyme; 0.32%). Total collection of milk, urine, and feces was conducted on d 19 to 21 of each pe-riod. There were no effects of dietary P or exogenous phytase plus cellulase on DMI, milk yield, or milk composition. Ex-cretion of feces was unaffected by diet, but urine excretion was less by cows fed the low P diets than by cows fed the high P diets (16.5 vs 21.3 kg/d). Com-pared with cows fed high P diets, cows fed the low P diets had reduced P intake (68.1 vs 103.9 g/d), reduced fecal (34.4 vs 51.3 g/d) and urinary P excretion (2.8 1 To whom correspondence should be ad-dressed: Knowlton@vt.edu vs 9.2 g/d), and lesser P balance (−8.0 vs. 4.4 g/d). The addition of exogenous phytase plus cellulase did not affect P in-take, milk P, fecal P, or urinary P excre-tion, but apparent P digestibility tended to be greater in cows fed diets supple-mented with the enzyme formulations (50.1% vs 40.5% for low P-enzyme and low P, respectively).
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ABSTRACT: One approach to reduce nutrient losses from livestock farms is to apply biological waste treatment systems such as biological nitrogen (N) removal or enhanced biological phosphorus (P) removal (EBPR) to reduce the nutrient content of land-applied waste. The EBPR process takes advantage of the ability of P-accumulating organisms (PAOs) to sequester excess P as polyphosphate granules in their cytoplasms, yielding a P-depleted liquid effluent and a P-enriched biomass. Biological N removal systems result in the conversion of organic or ammonia-N to innocuous N 2 gas. Understanding the variation in parameters such as chemical oxygen demand (COD), total and volatile suspended solids (TSS and VSS), and ammonia-N (NH 3 -N) is necessary to design these systems. Our objectives were to evaluate the effects of diet and manure separation on parameters important to reactor design. Waste was collected from nine cows fed a high P diet (0.47% P), a low P diet (0.32% P), or low P with exogenous phytase plus cellulase (0.32% P), in a replicated Latin square design (three 3 × 3 squares). Total collection of milk, urine, and feces was conducted on days 19 to 21 of each period, a mixed slurry (urine, feces, and water) was created, and slurry was separated mechanically to generate liquid effluent. Slurry contained more COD, solids, N, and P than liquid effluent, but the COD:P ratio was similar in the two wastes. The ratio of COD:N was higher in slurry than in separator effluent, but the ratio in both wastes was sufficient to support biological N removal. The P content of slurry, liquid effluent, and manure solids from cows fed low P was lower than from cows fed high P, and the COD content of effluent was higher with the low P diet. The COD:P ratio of all wastes was sufficient to support EBPR and biological N removal, but variation was observed with diet. Waste from cows fed low P had a higher COD:P ratio than that of cows fed high P, and waste from cows fed the enzyme-supplemented diet had a lower COD:N ration than that of cows fed the control diet. Dairy manure slurry and effluent will support EBPR and biological N removal. Dietary effects on parameters important to the design of advanced waste treatment systems were observed, but were not of a magnitude that would affect reactor design. Keywords. Lactating cows, Manure treatment, Research-scale manure separation. he development of strategies to reduce the nitrogen (N) and phosphorus (P) content of land-applied livestock manure is an important aspect of long-term efforts to reduce nutrient pollution of water re-sources. Reducing the nutrient content of land-applied waste reduces potential nutrient losses, allows livestock producers to increase the rate of manure application to a fixed land base, and/or reduces the amount of land needed for spreading ma-nure. Approaches to reducing the nutrient content of waste include dietary nutrient management (refining diets to maxi-mize efficiency of utilization of consumed nutrients) and physical or biological nutrient removal systems. Physical separation of manure solids and liquids via gravity or screening reduces organic loading to the liquid treatment system, removes large particles that could plug or damage nozzles in the irrigation system used in land Article was submitted for review in September 2004; approved for publication by the Structures & Environment Division of ASAE in March 2005.01/2005;
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ABSTRACT: The objective of this study was to evaluate the effect of an exogenous phytase on in vitro residual phosphorus (P) concentration and performance of 30 weaned ¾ Dorper vs ¼ Pelibuey lambs (12.12±1.46 kg BW). In vitro treatments were: 0 and 0.12 mg phytase per g of sorghum, corn gluten meal (CGM), alfalfa hay and experimental diet (70% ground sorghum grain, 16.9% CGM, 12% alfalfa hay, 1.1% calcium carbonate). There were significant (P<0.05) differences in the residual P concentration (%) between 0 and 0.12 mg phytase at 24h of incubation, the values being 0.086 and 0.050 for ground sorghum grain, 0.259 and 0.119 for CGM, 0.365 and 0.240 for alfalfa hay, and 0.276 and 0.240 for the mixed diet with the corresponding 48h values of 0.054 and 0.048 for ground sorghum grain, 0.178 and 0.161 for CGM, 0.198 and 0.131 for alfalfa hay, and 0.237 and 0.211 for the mixed diet. For the performance trial, 30 lambs were allotted to three different groups using complete randomised design, and were fed the experimental diet supplemented with 0, 6 or 12 g t-1 phytase. Parameters recorded were ADG, DM intake, feed conversion (FC), apparent DM digestibility and faecal P excretion (FPE). Phytase supplementation did not change (P>0.05) ADG (251, 294 and 266 g/d), DMI (905, 1119 and 975 g/d), FC (4.06, 4.37 and 3.94). However, phytase addition increased DMD (72.34, 82.54 and 82.57%) and FPE (1.01, 1.09 and 1.26 g/d). It may be concluded that apparent DM digestibility as well as faecal excretion of phosphorus were affected when an exogenous phytase was added to a 70% sorghum grain diet, fed to weaned Dorper x Pelibuey lambs.ANIMAL NUTRITION AND FEED TECHNOLOGY 01/2014; 14:183-188. · 0.36 Impact Factor
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ABSTRACT: A multifunctional transgenic Lactobacillus with probiotic characteristics and an ability to degrade β-glucan and phytic acid (phytate) was engineered to improve nutrient utilization, increase production performance and decrease digestive diseases in broiler chickens. The Bacillus subtilis WL001 endoglucanase gene (celW) and Aspergillus fumigatus WL002 phytase gene (phyW) mature peptide (phyWM) were cloned into an expression vector with the lactate dehydrogenase promoter of Lactobacillus casei and the secretion signal peptide of the Lactococcus lactis usp45 gene. This construct was then transformed into Lactobacillus reuteri XC1 that had been isolated from the gastrointestinal tract of broilers. Heterologous enzyme production and feed effectiveness of this genetically modified L. reuteri strain were investigated and evaluated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the molecular mass of phyWM and celW was approximately 48.2 and 55 kDa, respectively, consistent with their predicted molecular weights. Endoglucanase and phytase activities in the extracellular fraction of the transformed L. reuteri culture were 0.68 and 0.42 U/mL, respectively. Transformed L. reuteri improved the feed conversion ratio of broilers from 21 to 42 days of age and over the whole feeding period. However, there was no effect on body weight gain and feed intake of chicks. Transformed L. reuteri supplementation improved levels of ash, calcium and phosphorus in tibiae at day 21 and of phosphorus at day 42. In addition, populations of Escherichia coli, Veillonella spp. and Bacteroides vulgatus were decreased, while populations of Bifidobacterium genus and Lactobacillus spp. were increased in the cecum at day 21.International Journal of Molecular Sciences 01/2014; 15(7):12842-12860. · 2.46 Impact Factor
The Professional Animal Scientist 21 (2005):212–216
Exogenous Phytase Plus Cellulase
and Phosphorus Excretion in
Lactating Dairy Cows
K. F. KNOWLTON*,1, C. M. PARSONS*, C. W. COBB†, and K. F. WILSON†
*Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061
†Animal Feed Technologies, LLC, Greeley, CO 80632
The objectives were to assess the ef-
fects of exogenous phytase plus cellulase
on P excretion in lactating cows. The ef-
fects of an exogenous phytase plus cellu-
lase mixture and dietary P content on P
partitioning and excretion were evaluated
in nine early lactation cows (mean = 27
d in milk); six of the cows were rumi-
nally cannulated. Cows were assigned to
treatments in replicated (three) 3 × 3
Latin squares, and each cow received
each treatment sequentially in three, 21-
d periods. Diets were 45% forage (all
corn silage) and included supplemental P
(high P; 0.47%), no supplemental P (low
P; 0.32%), or no supplemental P with ex-
ogenous phytase (low P-enzyme; 0.32%).
Total collection of milk, urine, and feces
was conducted on d 19 to 21 of each pe-
riod. There were no effects of dietary P or
exogenous phytase plus cellulase on
DMI, milk yield, or milk composition. Ex-
cretion of feces was unaffected by diet,
but urine excretion was less by cows fed
the low P diets than by cows fed the
high P diets (16.5 vs 21.3 kg/d). Com-
pared with cows fed high P diets, cows
fed the low P diets had reduced P intake
(68.1 vs 103.9 g/d), reduced fecal (34.4
vs 51.3 g/d) and urinary P excretion (2.8
1To whom correspondence should be ad-
vs 9.2 g/d), and lesser P balance (−8.0
vs. 4.4 g/d). The addition of exogenous
phytase plus cellulase did not affect P in-
take, milk P, fecal P, or urinary P excre-
tion, but apparent P digestibility tended
to be greater in cows fed diets supple-
mented with the enzyme formulations
(50.1% vs 40.5% for low P-enzyme and
low P, respectively).
(Key Words: Phosphorus Excretion,
Phytase, Lactating Cows.)
The development of nutritional
strategies to reduce P excretion by
livestock is an important aspect of
long-term efforts to reduce P loading
to surface water. The availability of P
in feedstuffs affects P excretion, but
assumptions of availability of feed P
are based on relatively few studies
(Young et al., 1966; Dayrell and Ivan
1989; Martz et al., 1990; Martz et al.,
1999). Improved P availability from
feed would allow the tissue-level
needs of the animal to be met with
reduced P intake, thus reducing the P
content of livestock manure. The de-
velopment of phytase additives for
monogastric animals is one example
of nutritional manipulation of P avail-
ability and excretion. The endoge-
nous phytase activity provided by ru-
minal microorganisms makes the P in
grains and forages more available to
ruminants than to non-ruminants
(Clark et al., 1986; Morse et al.,
1992b). Because P intake and excre-
tion are so tightly linked (Morse et
al., 1992a; Wu et al., 2000; Knowlton
and Herbein, 2002), even small im-
provements in availability of feed P
would reduce P excretion signifi-
cantly. For instance, improving P
availability of dairy rations by 5 per-
centage units (i.e., from 60 to 65%)
and reducing P intake accordingly to
keep absorbed P constant would re-
duce P excretion by dairy cows by
15%, thus reducing the potential load-
ing to surface water significantly
(Knowlton et al., 2004).
There is some evidence in the litera-
ture that the endogenous phytase ac-
tivity of the ruminal microorganisms
varies with diet. Yanke et al. (1998)
observed that, in vitro, strains of Sele-
nomonas ruminantium, a starch-digest-
ing bacterium, had substantial phy-
tase activity and that phytase activity
of mixed ruminal fluid was greater in
steers fed a barley-based diet than in
those fed an all-hay diet. In a continu-
ous culture fermenter utilizing rumi-
nal fluid from goats, Godoy and
Meschy (2001) observed an increase
in phytate P availability with an or-
ganic P buffer compared with an inor-
ganic P buffer. Guyton et al. (2003)
observed an interaction of starch
source and supplementation with pu-
rified phytic acid on endogenous ru-
Exogenous Phytase and Phosphorus Excretion
minal phytase activity. The direction
of the response to phytic acid supple-
mentation differed with starch
source. In cows fed diets containing
dried ground corn, ruminal phytase
activity was numerically greater in
cows fed phytic acid than in cows fed
the low P diet. In contrast, in cows
fed diets based on steam-flaked corn,
phytase activity was similar with and
without phytic acid supplementation
(Guyton et al., 2003).
These studies, which indicate varia-
tion in endogenous ruminal phytase
activity, suggest opportunity to im-
prove P availability with exogenous
phytase under specific dietary condi-
tions. The objective was to evaluate
the effects of exogenous phytase plus
cellulase on P excretion in lactating
Materials and Methods
Cows and Diets. Nine early lacta-
tion cows (six ruminally cannulated;
27.2 ± 10.4 d in milk) were fed diets
containing 0.32 or 0.47% P (∼70 and
120% of NRC, 2001). The low P diets
were fed with or without the addi-
tion of fibrolytic and phytase enzyme
formulations. The fibrolytic enzyme
formulation was a commercial prepa-
ration from fungal extracts, with
15,000 units of cellulase activity/g
(Animal Feed Technologies, Greeley,
CO); one unit was defined as the cel-
lulase activity that produced a relative
fluidity change of 1.0 in 100 min in
a 0.2% (wt/vol) sodium carboxymthyl
cellulose (CMC type 7HP; Hercules,
Inc., Wilmington, DE) solution under
assay conditions (pH 4.5 and 40°C) as
measured with a Size 100 Calibrated
Cannon-Fiske type viscosimeter. The
phytase formulation contained 5000
units of phytase activity/g; one was
unit defined as the phytase activity
that released 1.0 —mol of phosphate/
min under assay conditions (pH 5.5
The granular enzyme formulations
were mixed with a corn grain carrier,
and the enzyme-corn grain mixture
or control (an equal quantity of corn
grain containing no enzyme formula-
TABLE 1. Ingredient composition of diets.
ItemHigh PLow PLow P-enzyme
(% of diet DM)
Expeller soybean meala
aSoyplus™ (West Central Soy, Ralston, IA).
bEach kilogram contained 1462 mg of Ca, 1758 mg of P, 11,100 mg of Mg, 1270
mg of S, 3.45 mg of Co, 136 mg of Cu, 6.8 mg of I, 170 mg of Mn, 2.64 mg of
Se, 340 mg of Zn, 46,900 IU of vitamin A, 16,960 IU of vitamin D, and 256 IU of
cEach tonne contained 200 g of fibrolytic enzyme formulation (15,000 units
cellulase activity/g) and 280 g of phytase (5000 units phytase activity/g).
tion) was added to the grain portion
of the diet prior to mixing of the
TMR (200 g of fibrolytic enzyme for-
mulation and 280 g of phytase/tonne
DM fed). Ingredient and nutrient
composition of diets are presented in
Tables 1 and 2. This experiment was
conducted with approval from the
Virginia Tech Animal Care Com-
Experimental Design and Sam-
pling. Cows were grouped by previ-
ous lactation mature-equivalent milk
yield and assigned to one of three, 3
× 3 Latin squares. Squares were bal-
anced for residual effects. Each experi-
mental period lasted 21 d. Cows were
fed in Calan doors for the first 17 d
of each period and were moved to in-
dividual stalls on d 18 for total collec-
tion of feces, urine, and milk. Cows
were fed once daily at 0800 h and
milked at 0700 and 1900 h. Feed was
offered at 5 to 10% in excess of previ-
ous day’s intake (wet basis).
On d 18, a sterile Foley urine cathe-
ter (22 French, 75 cc; C. R. Bard, Inc.,
Covington, GA) was inserted into the
urethra for total collection of urine.
All excreted urine, feces, and milk
were collected on d 19, 20, and 21.
Urine was weighed at 4-h intervals,
acidified to ph <2 (22 mL of 6N HCl/
kg urine), pooled, subsampled after
24 h, and stored frozen for later analy-
sis. All excreted feces were collected
at 4-h intervals and stored in a sealed
container, then weighed, thoroughly
mixed, and subsampled daily. Feed in-
gredients (forages and concentrates)
were sampled once each week, and
orts were weighed and sampled daily.
On d 19, 20, and 21, feed offered and
refused was measured, total milk
weights were recorded, and milk was
sampled at six consecutive milkings.
Laboratory Analysis. Samples of
feed ingredients and orts were dried
to constant weight at 60°C in a
forced-air drying oven (Wisconsin
Oven, Memmert; Schwabach, Ger-
many). Dried samples were ground
through a 1-mm screen in a Wiley
Mill (Arthur H. Thomas, Philadelphia,
PA). Feed and ort samples were ana-
lyzed in duplicate for N, P, Ca, ash
(AOAC, 1990), and NDF and ADF se-
quentially with α-amylase (Van Soest
et al., 1991). Feces and urine samples
were analyzed for P (AOAC, 1990)
and feces samples for NDF as indi-
cated previously. Milk samples were
Knowlton et al.
TABLE 2. Nutrient composition of diets.
Item High P Low PLow P-enzymeSEMTreatmentDietary Pa
(% of dietary DM)
aHigh P vs low P.
bLow P vs low P enzyme.
analyzed for fat, protein, total solids,
SNF (Dairy Herd Improvement Associ-
ation, Blacksburg, VA), and P (AOAC,
1990). Retention, milk output, and ex-
cretion of P were calculated.
Statistical Analysis. All data were
analyzed using the MIXED procedure
of SAS? (SAS Institute, Cary, NC)
with the model
Yijkl= µ + si+ cj(s)i+ Dk+Tl+ eijkl
µ = overall mean,
si= random effect of square
(i = 1 to 3),
TABLE 3. Effects of dietary P and addition of exogenous phytase plus cellulase on P intake and partitioning in
lactating Holstein cows.
ItemHigh PLow P Low P-enzymeSEM TreatmentDietary Pa
P Intake, g/d
Fecal P excretion, g/d
Apparent P digestibility, %
Urinary P, g/d
Total P excretion, g/d
Ruminal phytase activity,
nmol Pi released/min per mL rumen fluid
Milk P, g/d
Milk P, % of P intake
P Balance, g/d
aHigh P vs low P.
bLow P vs low P-enzyme.
cj(s)i= random effect of cow within
square (j = 1 to 3),
Dk= fixed effect of period
(k = 1 to 4),
Tl= fixed effect of treatment
(l = 1 to 3), and
eijkl= residual error.
Residual error was used to test the
main effect of treatment, and pre-
planned contrasts were used to evalu-
ate the effect of dietary P (high P vs
low P and low P-enzyme) and phy-
tase plus cellulase addition (low P vs
low P-enzyme). Differences were de-
clared significant at P < 0.05, and
trends were declared at P < 0.10 un-
less otherwise indicated. Results are re-
ported as least squares means.
Results and Discussion
Nutrient Composition of Diets. In-
gredient and nutrient composition of
treatment diets in Experiment 2 are
presented in Tables 1 and 2. As
planned, diets differed only in P
P Intake, Digestion, and Excre-
tion. Compared with cows fed high
P, cows fed the low P diets had re-
duced P intake (68.1 vs 103.9 g/d; Ta-
ble 3), reduced fecal (35.8 vs 51.3 g/d)
Exogenous Phytase and Phosphorus Excretion
TABLE 4. Effects of dietary P and addition of exogenous phytase plus cellulase on feed intake, digestibility, and
manure excretion in lactating Holstein cows.
ItemHigh P Low PLow P-enzyme SEMTreatmentDietary Pa
Apparent DM digestibility, %
Fecal excretion, kg/d DM
Fecal excretion, kg/d wet
Urine output, kg/d
aHigh P vs low P.
bLow P vs low P-enzyme.
and urinary P excretion (1.5 vs 5.4 g/
d), and lesser P balance (−6.7 vs 8.3 g/
d). Others have observed similar re-
duced fecal and urinary P excretion
with decreased dietary P intake (Wu
et al., 2000; Knowlton et al., 2001;
Wu et al., 2001; Knowlton and Herb-
ein, 2002). Apparent P digestibility
was unaffected by dietary P content,
similar to the observations of Guyton
et al. (2003). Milk P secretion as a pro-
portion of P intake was greater in
cows fed the low P diets than in cows
fed the high P diets (51.5% vs
34.9%). Morse et al. (1992a), Knowl-
ton et al. (2001), and Knowlton and
Herbein (2002) also observed that
milk P as a percentage of P intake de-
creased with an increase in dietary P
content, as the P content of milk is
TABLE 5. Effects of dietary P and addition of exogenous phytase plus cellulase on lactational performance in
lactating Holstein cows.
Item High P Low PLow P-enzymeSEM TreatmentDietary Pa
Milk yield, kg/d
Milk fat, kg/d
True protein, kg/d
Milk urea N, mg/dL
aHigh P vs low P.
bLow P vs low P-enzyme.
Addition of exogenous cellulose
and phytase did not affect P intake,
milk P, fecal P, or urinary P excretion
(Table 3), but apparent P digestibility
tended to be greater in cows supple-
mented with exogenous enzymes
(50.1% vs 40.5% for low P-enzyme
and low P, respectively; P<0.11). This
trend was due to a slight, non-signifi-
cant, increase in P intake (+2.8 g/d)
combined with a numerical decrease
in fecal P excretion (−5.4 g/d) with
phytase supplementation. Most pub-
lished studies have reported that ru-
minal phytase activity does not limit
digestion of dietary P (Reid and Frank-
lin, 1947; Clark et al., 1986; Morse et
al., 1992b). However, there is some ev-
idence of incomplete digestion of
phytic acid in ruminants. Hill et al.
(2002) reported that low phytic acid
corn, but not exogenous phytase, re-
duced fecal P concentration in midlac-
tation cows. Interpretation of that
study is problematic, as the low phy-
tic acid corn was not isogenic with
the normal corn. Duskova et al.
(2001) observed measurable phytic
acid in the feces of grain-fed, weaned
calves at 6 and 13 wk of age, indicat-
ing incomplete digestion.
An alternative explanation for the
effect of the supplementation with ex-
ogenous enzymes on apparent P di-
gestibility is increased digestion of
the entire diet caused by the cellulase
in the formulation. The lack of effect
of enzyme addition on apparent DM
digestibility (Table 4) does not sup-
port this explanation. Additional
work is needed to clearly separate any
effects of the two enzymes and to
Knowlton et al.
evaluate varying doses of exogenous
phytase in different basal diets.
Feed Intake, Manure Production,
and Milk Yield. Intake and digestibil-
ity of DM and excretion of feces were
unaffected by diet (Table 4), but urine
excretion was less by cows fed the
low P diets than by cows fed the
high P diet (16.5 vs 21.3 kg/d). Only
one other experiment has reported
an effect of dietary P content on
urine excretion. Burkholder et al.
(2004) observed that cows fed supple-
mental purified phytic acid excreted
more urine (+1.9 kg/d) than cows fed
low P diets. The effect of dietary P on
urine excretion is likely an indirect ef-
fect, but, in the absence of water con-
sumption data, the biological mecha-
nism is unclear. Diets were formu-
lated to contain the same Na and K
content, with the same quantities of
salt and sodium bicarbonate provided
to all cows.
Neither dietary P content nor exog-
enous phytase plus cellulase affected
milk yield (39.6 kg/d) or milk compo-
sition (Table 5). Milk fat content was
low (≤3% or less), reflecting the rela-
tively low forage content (45%) and
the use of corn silage as the sole
source of forage.
Reduced dietary P reduced P excre-
tion and improved capture of dietary
P in milk. Addition of exogenous phy-
tase plus cellulase to the low P diet
tended to improve apparent P digest-
ibility. Additional work is needed to
clearly separate the effects of the two
enzymes, to evaluate varying doses of
exogenous phytase in different basal
diets, and to evaluate effects of low P
diets with and without phytase on P
balance throughout lactation. Utiliza-
tion of exogenous enzyme formula-
tion in conjunction with reduced di-
etary P may improve producers’ abil-
ity to meet P-based nutrient
Financial support for this project
was provided by Animal Feed Techno-
logies LLC (Greeley, CO). The authors
appreciate the technical support pro-
vided by Harold Nester, Chuck Miller,
and William Saville. Assistance pro-
vided by Colin Albertyn, Gary Braun-
ing, Barnett Carr, Rebecca Cornman,
Angela Gamboni, Krystal Hardin, Ja-
neen Lewis, Christopher Lilly, Julie
McKinney, Catherine Parsons, Eric
Paulson, Jason Poston, Steven
Salmon, and Kristi Seat during the col-
lection periods and sample analysis is
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