1. 411
J. Dairy Sci. 97:411–418
http://dx.doi.org/10.3168/jds.2013-7124
© American Dairy Science Association®
, 2014.
ABSTRACT
Accurate estimates of phosphorus (P) availability
from feed are needed to allow P requirements to be
met with reduced P intake, thus reducing P excretion
by livestock. Exogenous phytase supplementation in
poultry and swine diets improves bioavailability of P,
and limited research suggests that this strategy may
have some application in dairy cattle rations. The ef-
fects of exogenous phytase and forage particle length
on site and extent of P digestion were evaluated with
5 ruminally and ileally cannulated lactating cows
(188 ± 35 d in milk). Cows were assigned in a 2 ×
2 factorial arrangement of treatments in 2 incomplete
Latin squares with four 21-d periods. Diets contained P
slightly in excess of National Research Council require-
ments with all P from feed sources. During the last 4
d of each period, total mixed ration, refusals, omasal,
ileal, and fecal samples were collected and analyzed for
total P, inorganic P (Pi), and phytate (Pp). Total P
intake was not influenced by dietary treatments but Pp
intake decreased and Pi intake increased with supple-
mental phytase, suggesting rapid action of the enzyme
in the total mixed ration after mixing. Omasal flow
of Pi decreased with phytase supplementation, but we
observed no effect of diet in ileal flow or small intestinal
digestibility of any P fraction. Fecal excretion of total
P was slightly higher and Pp excretion was lower for
cows receiving diets supplemented with phytase. Milk
yield and composition were unaffected by diets. When
phytase was added to the mixed ration, dietary Pp
was rapidly degraded before intake and total-tract Pp
digestion was increased. The lack of effect of phytase
supplementation on dietary P utilization was probably
because these late-lactation cows had a low P require-
ment and were fed P-adequate diets.
Key words: phytase, phytate digestion, lactating
dairy cow
INTRODUCTION
Dietary manipulation to reduce phosphorus (P)
excretion by dairy cattle is an important approach to
optimize whole-farm nutrient balance and minimize
environmental P pollution from dairy farms (Morse et
al., 1992; Wu et al., 2001). Phytate is approximately
70% of the total P in grains (Nelson et al., 1968) and
although ruminal microorganisms have the capacity to
liberate phosphate from the inositol ring of the phytate
molecule (Clark et al., 1986; Morse et al., 1992), the
degradation of phytate P (Pp) in the rumen may not
be complete (Godoy and Meschy, 2001). Exogenous
phytase supplementation has been reported to reduce
fecal P excretion in lactating cows (Kincaid et al., 2005;
Knowlton et al., 2007). Ruminal Pp degradation is in-
fluenced by digesta passage rate, physical properties of
the ration, and grain type (Kincaid et al., 2005) and,
in sheep, feed processing alters Pp digestion (Bravo et
al., 2000; Park et al., 2000). Variation in availability of
Pp is not accounted for in current ration formulation
programs for dairy cows.
Digesta passage rate increases with decreasing forage
particle length in ruminants (Rode et al., 1985). Faster
passage rate due to finely chopped diets may limit ru-
minal phytate degradation in dairy cows because of the
short-duration exposure of phytate molecule to micro-
bial phytase. Ruminal phytate degradability decreased
from 84% to 69 and 57% when ruminal passage rate
increased from 0.02/h to 0.05 and 0.08/h, respectively
(Park et al., 1999). This limitation could be resolved
if sufficient postruminal phytate degradation is pos-
sible. Phytase activity depends on pH, and optimum
phytase activity is at low pH (pH 4–5.5; Yanke et al.,
1999). The proximal small intestine can serve as the
primary site of postruminal phytate degradation be-
cause of its acidic pH. We hypothesized that ruminal
phytate degradation would be incomplete in cows fed
short-particle-length forage and that supplementation
of rumen-protected phytase would compensate for this
by releasing inorganic P from the phytate molecule in
the proximal small intestine. Therefore, the objective of
this study was to investigate site and extent of Pp deg-
radation in lactating cows fed diets varying in forage
The effects of forage particle length and exogenous phytase inclusion
on phosphorus digestion and absorption in lactating cows
J. P. Jarrett, J. W. Wilson, P. P. Ray, and K. F. Knowlton1
Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061
Received June 12, 2013.
Accepted September 2, 2013.
1
Corresponding author: knowlton@vt.edu

2. 412 JARRETT ET AL.
Journal of Dairy Science Vol. 97 No. 1, 2014
particle length with and without exogenous ruminally
protected phytase.
MATERIALS AND METHODS
Animals and Diets
Five crossbred [Swedish Red or Brown Swiss ×
(Holstein × Jersey)] ruminally and ileally cannulated
first-lactation cows (BW of 472 ± 36 kg and 188 ± 35
DIM) were utilized in 2 incomplete 4 × 4 Latin squares.
Eight cannulated cows were surgically prepared 2 yr
before this experiment; in the interim, 2 lost their ileal
cannula. To take full advantage of the statistical power
offered by 6 cows, two 4 × 4 Latin squares balanced
for carryover effects were established; the 8 treatment
sequences (4 per square) were distinct. One row in each
square was randomly selected for removal to yield 2
incomplete Latin squares. Six cows were then randomly
assigned to square and row (treatment sequence). One
of the 6 cows died unexpectedly before the study began
of an unrelated health problem. In the resulting 5-cow
experiment, each cow received each treatment one time,
and carryover effects were balanced within square.
The treatments, inclusion of phytase (1,500 FTU/kg
of diet DM) and forage particle length (short vs. long),
were administered in a 2 × 2 factorial arrangement.
Diets were otherwise identical in composition (Table
1) and formulated to meet or exceed NRC (2001) re-
quirements for dairy cows. Dietary P concentration was
0.43% of DM. Particle length treatments were long for-
age or short forage, achieved by differential processing
of grass hay and corn silage. Grass hay was cut to 6.35
and 0.64 cm using a tub grinder (New Holland 390, Ra-
cine, WI) for long forage and short forage treatments,
respectively, with hay sufficient for the duration of the
study cut in advance. Theoretical length of cut for
corn silage at harvest was 0.95 cm, and corn silage for
SF was passed through a leaf mulcher (Flowtron Leaf
Eater, Malden, MA) on the fine setting daily before
mixing the experimental diets. Phytase was added to
the vitamin-mineral mix premade in batches sufficient
for 1 period. Corn silage, grass hay, grain mix, and
vitamin-mineral mix (with or without phytase) were
mixed daily at 1200 h to prepare the experimental
TMR.
When cows were not in the metabolism stalls for total
collection (described below), they were group-housed in
a freestall barn and fed once daily via the Calan door
system (American Calan, Northwood, NH). Cows were
fed 10% excess of the previous day’s intake at 1230 h,
and feed refusals were measured daily. Milk yield was
measured twice daily at each milking, at 0600 and 1800
h. All protocols and procedures were approved by the
Virginia Tech Institutional Animal Care and Use Com-
mittee (IACUC #10-105 DASC).
Experimental Design and Sampling
Each of four 21-d periods consisted of 14 d of dietary
adaptation in freestalls, 3 d of adaptation to metabo-
lism stalls, and 4 d of total collection. Lithium cobalt
(Co)-EDTA solution (Uden et al., 1980) and ytterbium
(Yb)-labeled corn silage (Harvatine et al., 2002) were
used as liquid and solid phase markers, respectively, to
measure digesta flow. Immediately before each feeding,
markers were dosed ruminally to supply 0.11 g/d each
of Co and Yb. Cows were milked twice daily at 0600
and 1800 h, and milk weights were recorded at each
milking. Milk samples were collected at each milking
and stored at −20°C for future analysis. Beginning on
d 17 of each period, cows were fed 25% of their daily
feed allowance 4 times daily at 0600, 1200, 1800, and
2400 h to reduce diurnal variation in intake and diges-
tion. On d 16, 24 h before the onset of total collection,
cows were fitted with harnesses that linked to a cup
covering the vulva (Fellner et al., 1988) for total daily
urine collection into a 12-L jug that was maintained on
ice (Knowlton et al., 2010). Beginning at 1800 h on d
17, feces were collected from behind each cow 4 times
daily and stored in a 130-L tub. At 1800 h on d 18, 19,
20, and 21, feces, urine, and refusals were weighed, and
homogeneous subsamples were collected and stored at
−20°C until further analysis.
Table 1. Ingredient and nutrient composition of diets
Item
% of
DM
Ingredient
Corn silage 41.7
Grass hay 13.5
Cottonseed meal 15.6
Corn, ground 12.3
Soybean meal, 48% 7.9
Molasses, dehydrated 3.5
Beet pulp, dehydrated 2.9
Vitamin-mineral mix1
1.9
Sodium bicarbonate 0.6
Nutrient
DM 58.5
CP 17.7
NDF 32.6
ADF 18.8
Ca 0.66
P 0.43
1
Vitamin-mineral mix contains vitamin A 26,400 kIU/kg, vitamin D
8,800 kIU/kg, vitamin E 44,000 IU/kg, 7.6% Ca, 5.9% Cl, 4.7% Na,
0.85% S, 0.45% Mg, 0.03% K, 3,500 mg/kg Zn, 2,000 mg/kg Fe, 2,000
mg/kg Mn, 300 mg/kg Cu, 90 mg/kg Se, 70 mg/kg I, and 50 mg/kg
Co.

3. Journal of Dairy Science Vol. 97 No. 1, 2014
EFFECTS OF FORAGE PARTICLE LENGTH AND EXOGENOUS PHYTASE 413
At 1400 h on d 16 (the evening before total collec-
tion commenced), a sampler was placed into the omasal
orifice for sampling, as described by Huhtanen et al.
(1997). Omasal and ileal samples were collected every
8 h on d 18 to 21 advancing by 2 h on each consecutive
day to represent every 2 h of a 24-h period.
Coccygeal blood samples were collected into serum
separator tubes (Fisher Scientific, Pittsburgh, PA) on
d 18 through 21 before the 1200 h feeding. Upon collec-
tion, samples were immediately centrifuged (2,200 × g
for 20 min at 5°C), and serum was harvested and stored
at −20°C until further analysis. Rumen evacuation was
conducted on d 21 of each period. Rumen contents were
weighed and representative samples were collected be-
fore returning the contents to the rumen.
Sample Analysis
Total mixed ration particle size distribution was
determined using the Penn State Particle Separator
(Kononoff et al., 2003). Frozen samples of feed, refus-
als, omasal, ileal, and fecal samples were thawed and
composited by wet weight by cow × period. Samples of
feed and refusals were oven-dried in a 55°C forced-air
oven (Thermo Scientific Precision 645, Danville, IN)
to a constant weight. Rumen, ileal, and fecal samples
were lyophilized (Genesis 25EL, Stone Ridge, NY) and
ground through a 1.0-mm screen in a Wiley mill (Ar-
thur H. Thomas, Philadelphia, PA) and then through
a Z grinder (0.2-mm screen; Retsch ZM 100, Haan,
Germany). Because of their heterogeneous nature, wet
omasal samples were separated to liquid and solid frac-
tions and ground in a freezer mill (described below). All
samples were analyzed in duplicate for N, P (AOAC,
1984; method 965.17), NDF (Mertens, 2002), and ADF
sequentially (Van Soest et al., 1991), and for Ca using
inductively coupled plasma atomic emission spectros-
copy (Thermo Electron IRIS Intrepid II XSP; Thermo
Fisher Scientific Inc., Waltham, MA). Milk samples
were analyzed for fat, protein, SNF, lactose, MUN, and
SCC (DHIA, Blacksburg, VA).
Thawed composite omasal samples were centrifuged
at 1,000 × g for 5 min to separate flow into 2 phases;
fluid and particulate (the latter containing small and
large particles; adapted from Reynal and Broderick,
2005). Both phases were frozen at −80°C and then ly-
ophilized (Genesis 25EL). The fluid phase was ground
using a freezer mill (SPEX 6850 Freezer Mill, SPEX
CertiPrep Inc., Metuchen, NJ) and the particulate
phase was ground through the 0.2-mm screen in a Z-
grinder (Retsch ZM 100). Both phases were analyzed
for Co and Yb by inductively coupled plasma atomic
emission spectroscopy (Thermo Electron IRIS Intrepid
II XSP; Thermo Fisher Scientific Inc.) and data were
used to define relative omasal flow of fluid and par-
ticulates. Freeze-dried fluid and particulate phases
were recombined in proportions to their relative con-
tribution to true omasal DM flow using the 2-marker
system (France and Siddons, 1986). Omasal, ileal, and
fecal samples were analyzed for Co and Yb; data from
the first 2 were used to calculate nutrient flow, and
fecal data were used to calculate actual dose of each
marker for each day. Urine samples were analyzed for P
(AOAC, 1984; method 965.17). Serum inorganic P (Pi)
was determined using colorimetric methods (Inorganic
Phosphorus Reagent Set, Pointe Scientific, Canton,
MI).
Phosphorus Fractionation
For Pi analysis, 1 g of dry sample (TMR, refus-
als, digesta, feces) was extracted with 20 mL of 0.5
M HCl by shaking at ambient temperature (25°C) on
a horizontal mechanical shaker (Eberbach Reciprocal
Shaker 6000, Ann Arbor, MI) for 4 h. After shaking,
the samples were refrigerated (4°C) overnight. Samples
were then centrifuged at 30,000 × g for 10 min at 4°C.
Supernatants were collected to analyze for Pi using the
molybdenum blue method as described by Murphy and
Riley (1958).
Samples were extracted and analyzed for phytate
and lower inositol phosphates as described by Ray et
al. (2012a,b). Briefly, samples were extracted in dupli-
cate with either 0.5 M HCl (feed and refusals) or 0.25
M NaOH-50 mM EDTA (digesta and feces). Alkaline
extracts of digesta and feces were acidified with 6 M
HCl-1.2 M HF acid reagent (500 μL of acid reagent into
5 mL of extract) and stored overnight at 4°C. Acidified
and refrigerated extracts were centrifuged at 30,000 ×
g for 10 min at 4°C and clear supernatants were col-
lected. Supernatants were passed through a methanol-
conditioned C18 cartridge (Sep-Pak Plus, Waters, MA)
and a 0.2-μm ion chromatography membrane filter.
Filtered feed extracts were analyzed by high perfor-
mance ion chromatography (HPIC; Dionex ICS 3000
with a Dionex 4 × 50 IonPac AG7 guard column and
a 4 × 250 mm IonPac AS7 analytical column; Dionex,
Sunnyvale, CA) using an isocratic 10-min elution meth-
od with 0.25 M HNO3 as the mobile phase. For digesta
and feces extracts, a pH 4, 20.1-min gradient elution
(0.01 M methylpiperazine and 0.01 M NaNO3-0.01 M
methylpiperazine) method was used. In feed extracts,
phytate was detected using a UV-visible detector with
absorbance monitored at 290 nm following postcolumn
colorimetric reaction with 0.1% Fe(NO3)3 in 2% HClO4.
For phytate and lower inositol phosphate detection in
feces and digesta extracts, Wade’s reagent (0.015%
FeCl3 + 0.15% sulfosalicylic acid solution) was used for

4. 414 JARRETT ET AL.
Journal of Dairy Science Vol. 97 No. 1, 2014
postcolumn derivatization, and absorbance wavelength
was 500 nm (Rounds and Nielsen, 1993).
Statistical Analysis
All data were analyzed using the mixed procedure
of SAS (SAS Institute Inc., Cary, NC) with a model
including square, period, phytase, forage particle
length, and the interaction of phytase and forage par-
ticle length as fixed effects, and cow within square as
random effect (Table 2). Somatic cell count data were
log-transformed before statistical analysis. The residual
tested the main effects, and cow within square tested
the effects of square. Significant differences were de-
clared at P < 0.05 and trends at P < 0.15. Results are
reported as least squares means.
RESULTS AND DISCUSSION
Effect of Grinding on Particle Size
We observed a reduction in the proportion of par-
ticles >19 mm and particles of 8 to 1.18 mm with the
SF diet and a tendency (P = 0.06) for the SF rations
to have more fine particles (particles <1.18 mm; Table
3). Thus, chopping the forage effectively reduced the
forage particle length in the SF rations.
DMI and Digestibility
Dry matter digestibility tended to decrease (P =
0.08) with short forage (Table 4), as has been reported
previously (Rode et al., 1985; Zebeli et al., 2007), likely
due to a more rapid passage rate. Dry matter intake
was not influenced by forage particle length (Table 4).
Similarly, forage particle length did not influence DMI
in early-lactation cows fed TMR containing 40% alfalfa
silage (Krause et al., 2002). Beauchemin et al. (2003)
observed that the effect of forage particle size on DMI
may depend on amount of dietary forage, but Yang
and Beauchemin (2007) did not observe any effect of
forage particle length on DMI regardless of dietary
forage:concentrate ratio (35:65 vs. 60:40). Voelker et al.
(2002) reported that limitation of DMI by rumen-fill
diets is more prominent in high-producing cows than
in low-producing cows, so the lack of response in DMI
in this study may be due to low production at the late
stage of lactation.
Milk Production
Effect of Forage Particle Length. Forage particle
length did not influence milk yield or milk components
(Table 5), similar to results observed by Krause et al.
(2002) and Beauchemin et al. (2003) in corn silage- or
alfalfa-based forage diets, respectively. Similarly, yields
of milk and milk components were not influenced by
forage particle length in late lactation Holstein cows
fed an orchardgrass silage-based 50% forage diets
(Kammes and Allen, 2012). In the current study, the
lack of response in milk yield to particle size is likely
the reflection of unchanged DMI and total-tract DM
digestibility, because forage particle length primarily
influences milk production via change in DMI (Krause
et al., 2002). Lack of response in milk fat yield was due
to the presence of sufficient dietary fiber to maintain
milk fat production (Mertens, 1997).
Effect of Phytase. Milk production, 3.5% FCM,
and SCS were similar in cows receiving diets with or
without phytase (Table 5). Yields of protein, fat, SNF,
and lactose were not affected by phytase supplementa-
tion. Similarly, supplemental phytase did not influence
the yield of milk and milk components in late-lactation
dairy cows, irrespective of dietary grain source (Kin-
caid et al., 2005). In nonruminant animals, it is very
Table 2. Statistical model used to analyze all variables
Source df
Error
df
Error
term
Square 1 3 Cow(square)
Cow(square) 3
Phytase 1 9 Residual
Particle length 1 9 Residual
Phytase × particle length 1 9 Residual
Period 3 9 Residual
Residual 9
Total 19
Table 3. Effect of hay and corn silage processing on particle size of TMR samples measured with the Penn
State Particle Separator
Particle size,
mm
No phytase Phytase
SEM
P <
Short Long Short Long Phytase FPL1
Phytase × FPL
>19 2.1 12.8 2.3 12.9 1.2 0.92 0.01 0.98
19–8 32.3 33.1 28.7 31.2 2.1 0.31 0.33 0.87
8–1.18 45.4 34.6 47.3 38.4 2.2 0.23 0.01 0.69
<1.18 21.2 19.4 21.7 17.4 1.4 0.60 0.06 0.40
1
Forage particle length.

5. Journal of Dairy Science Vol. 97 No. 1, 2014
EFFECTS OF FORAGE PARTICLE LENGTH AND EXOGENOUS PHYTASE 415
common to observe improvements in growth and pro-
duction with dietary phytase addition or by feeding low
phytate diets (Kemme et al., 1999; Bohlke et al., 2005;
Liao et al., 2005). These positive responses in growth
and production may stem from an improvement in the
digestibility of N and amino acid, and(or) increased
metabolizable energy with phytase supplementation
(Ravindran et al., 2001). In the present study, the lack
of response in milk and milk component yields may
have been due to reduced demand for protein and ener-
gy with relatively low milk production in late lactation.
Even in early-lactation cows, however, effects of phy-
tase on production have not been observed. Knowlton
et al. (2005) reported no difference in milk yield or
component production for early-lactation cows (27 ±
10 DIM) fed a phytase-cellulase enzyme blend supple-
mented diet and no effect on these measures in a second
study with the same phytase blend fed to Holsteins
averaging 95 DIM and 38 kg/d milk (Knowlton et al.,
2007). Therefore, we speculate that the lack of exog-
enous phytase effect on milk production may be due
to sufficient intrinsic microbial phytase activity in the
rumen to improve nutrient availability, even in the con-
trol cows.
Phosphorus Intake, Flow, Excretion, and Digestibility
Effect of Forage Particle Length. Forage particle
length did not influence intake and omasal flow of total
P or Pi. Ruminal pool size (Table 6) and digestibility of
total P, Pi, and Pp were not affected by forage particle
length. Flow of total P and Pi at the ileum was in-
creased with short forage, possibly because of increased
postruminal digesta passage rate, but ileal flow of Pp
was not influenced by forage particle length (Table 7).
Fecal excretion of total P increased slightly with short
forage (57.6 vs. 54.3 g/d; Table 7), perhaps a result of
increased passage rate implied by the tendency of de-
creased DM digestibility with short forage and reported
by others (Yang and Beauchemin, 2007).
Recycling of endogenous P via salivary P secretion
is a salient physiological characteristic of ruminants.
Long forage particle length can increase salivation rate,
which in turn may influence net loss of endogenous P.
However, we did not expect any influence of salivary
P on endogenous P in this experiment because when
diet composition, DMI, and milk yield are not different,
salivary P concentration is regulated by P intake (Valk
et al., 2000). In this experiment, late-lactation cows
were provided the same P-adequate diets, and dietary
treatments did not influence milk production, DMI, or
P intake. Serum P was >5 mg/dL, which is considered
normal in lactating cows (Forar et al., 1982), and did
not differ between the treatment groups. In ruminants,
salivary P concentration is positively correlated with
blood P concentration and has an inverse relationship
with saliva flow rate (Cohen, 1980; Valk et al., 2002;
Block et al., 2004). Therefore, adequate dietary P and
Table 4. Effect of particle size (short or long) and phytase on DMI, DM digestibility, and fecal and urinary
output
Item
No phytase Phytase
SEM
P <
Short Long Short Long Phytase FPL1
Phytase × FPL
DMI, kg/d 16.1 17.1 17.0 17.2 0.57 0.35 0.23 0.43
DM digestibility,2
% 66.0 69.2 66.0 67.8 1.27 0.56 0.08 0.58
Feces, kg of DM/d 5.4 5.3 5.7 5.6 0.25 0.27 0.65 0.82
Urine, kg/d 11.5 10.8 10.3 11.6 1.25 0.80 0.71 0.23
1
Forage particle length.
2
Apparent total-tract digestibility.
Table 5. Effect of particle size (short or long) and phytase on milk production and composition
Item
No phytase Phytase
SEM
P <
Short Long Short Long Phytase FPL1
Phytase × FPL
Milk, kg/d 19.5 20.3 20.2 20.9 0.78 0.38 0.34 0.92
Fat, kg/d 1.00 1.00 1.05 1.07 0.04 0.20 0.82 0.88
Protein, kg/d 0.77 0.79 0.82 0.81 0.03 0.23 0.83 0.68
SNF, kg/d 1.89 1.97 1.99 2.04 0.07 0.29 0.45 0.84
Lactose, kg/d 0.96 1.00 1.00 1.04 0.04 0.33 0.31 0.96
MUN, mg/dL 11.5 11.0 10.8 11.7 0.63 0.97 0.63 0.18
SCS 1.76 1.68 1.65 1.54 0.18 0.18 0.32 0.82
1
Forage particle length.

6. 416 JARRETT ET AL.
Journal of Dairy Science Vol. 97 No. 1, 2014
no difference in P intake and blood P concentration
corroborate the conclusion that salivary P did not in-
fluence net endogenous P loss and subsequently had no
effect on the results of this experiment.
Effect of Phytase. As intended, exogenous phytase
had no effect of on total P intake, and serum, urinary,
and retained P were not influenced by phytase (Table
7). Kincaid et al. (2005) reported an increase in serum
P in lactating cows fed exogenous phytase in barley-
and corn-based diets, but most reports show changes
in serum P only when comparing P-adequate with P-
deficient diets (Wu et al., 2001; Knowlton and Herbein,
2002).
Phytase inclusion decreased Pp intake (18.4 vs.
40.7 g/d) and increased Pi intake (46.3 vs. 34.1 g/d;
Table 7). This shift in dietary P form with inclusion
of exogenous phytase indicates rapid phytase activity
at the time feed was mixed. Samples were collected at
mixing and were frozen within 45 min. The ambient
temperature (midday temperatures from 13 to 28°C)
and water content (41.5% moisture) of the mixed ration
were conducive to rapid phytase activity (Poulsen et
al., 2012). Kincaid et al. (2005) fed exogenous phytase
to lactating dairy cows, but observed no change in di-
etary Pp content; relative timing of mixing, sampling,
and storage were not reported.
Omasal flow of Pi tended to decrease with phytase
inclusion (148.3 vs. 178.0 g/d; Table 7), possibly indi-
cating increased conversion of the Pi to microbial P in
the rumen because more Pi was immediately available
in cows fed phytase-amended diets. Subsequent flow of
Pi to the ileum and fecal excretion of Pi were unaffected
by phytase, and relative flows of Pi (omasal dietary
> ileal = fecal) were as expected. Approximately 70 to
80% of omasal Pi was absorbed in the small intestine,
and net disappearance of Pi in the large intestine was
not different from zero.
Phytate flow at the omasum was approximately 1
g/d (95% CI: 0.52 to 1.39 g/d) and was slightly greater
at the ileum (95% CI: 1.36 to 1.84 g/d) than at the
omasum with exogenous phytase. This biologically un-
likely observation is probably because these samples
were very near the limit of quantification of our analyti-
cal methods. Also, the very high Pi content and low Pp
Table 6. Effect of forage particle length and phytase on rumen nutrient pool size
Item
No phytase Phytase
SEM
P <
Short Long Short Long Phytase FPL1
Phytase × FPL
DM, kg 8.4 9.1 8.9 9.4 0.77 0.63 0.48 0.93
Total P, g 65.4 71.9 63.9 67.5 5.8 0.58 0.35 0.78
Inorganic P, g 41.7 51.8 44.9 51.6 5.1 0.77 0.12 0.73
1
Forage particle length.
Table 7. Effect of forage particle length and phytase on intake, omasal flow, ileal flow, and fecal excretion of
total P, inorganic P, and phytate P
Item
No phytase Phytase
SEM
P <
Short Long Short Long Phytase FPL1
Phytase × FPL
Intake
Total P, g/d 74.5 76.7 77.8 74.1 4.08 0.93 0.86 0.49
Inorganic P, g/d 32.8 35.5 49.2 43.5 3.19 0.01 0.60 0.16
Phytate P, g/d 44.0 37.3 13.8 23.1 3.88 0.01 0.74 0.07
Omasal flow
Total P, g/d 196 182 174 158 17.6 0.12 0.28 0.94
Inorganic P, g/d 181 175 160 137 16.9 0.06 0.27 0.56
Phytate P, g/d 0.87 0.94 0.99 1.10 0.55 0.73 0.83 0.97
Ileal flow
Total P, g/d 72.6 53.6 74.0 61.4 5.4 0.24 0.01 0.41
Inorganic P, g/d 43.4 33.4 49.0 36.3 6.9 0.21 0.01 0.68
Phytate P, g/d 2.03 1.58 1.67 1.54 0.28 0.42 0.25 0.51
Fecal excretion
Total P, g/d 54.7 50.4 59.4 55.3 2.35 0.02 0.04 0.93
Inorganic P, g/d 41.4 34.2 41.3 42.0 3.21 0.21 0.29 0.20
Phytate P, g/d 1.49 1.16 0.97 0.99 0.24 0.01 0.18 0.15
Urinary P, g/d 0.77 2.62 0.99 1.70 1.08 0.68 0.15 0.50
Serum P, mg/dL 5.41 5.93 5.33 5.64 0.46 0.59 0.25 0.75
Retained P, g/d −0.5 3.3 −2.8 −3.9 3.7 0.22 0.71 0.51
1
Forage particle length.

7. Journal of Dairy Science Vol. 97 No. 1, 2014
EFFECTS OF FORAGE PARTICLE LENGTH AND EXOGENOUS PHYTASE 417
content of omasal samples present challenges to HPIC
analysis. Large orthophosphate peaks make it difficult
to differentiate between Pp peaks and noise.
To fully account for effects of phytase, total-tract Pp
digestibility was calculated assuming similar original
(at mixing) Pp content of treatment diets with and
without phytase. This measure indicated that phytase
increased Pp digestibility from 96.7 to 97.6%. Kincaid
et al. (2005) observed increased total-tract Pp hy-
drolysis with phytase supplementation (85 vs. 80%).
The much greater fecal excretion of Pp observed by
Kincaid et al. (2005; 10 and 14 g/d with and without
phytase addition, respectively, compared with ~1 g/d
of Pp in the present study) is due to dietary differences
as well as higher DMI in the former study compared
with the cows in the present study. Also, Kincaid et al.
(2005) detected phytate (inositol hexaphosphate) and
the lower inositol phosphates collectively as phytate
because they used the ferric precipitation method with
less separation capacity than the HPIC method used in
the present study.
Fecal excretion of total P increased with phytase
inclusion, a small and not easily explained observation.
Fecal Pp excretion was slightly (but significantly) lower
with dietary phytase (Table 7). Although the values of
ileal and fecal Pp flow were small, observed values were
significantly different from zero.
Net disappearance of Pp from the large intestine was
observed, with 25 to 40% of Pp entering the ileum dis-
appearing from the large intestine. Park et al. (2002)
observed slightly lower relative large intestine digest-
ibility of Pp (~19%) in sheep, as did Ray et al. (2012b,
2013) in dairy heifers and dairy cows (15–16%). Large-
intestine Pp hydrolysis is due to enzymatic activity of
phytase produced by large intestinal microbial popula-
tion (Wise and Gilburt, 1982; Matsui et al., 1999) and
would be of nutritional significance if the animal can
absorb digested P from the large intestine. Large intes-
tinal disappearance of total P was significantly different
from zero and up to 25% of ileal flow. This is within the
range of net disappearance of P from the large intestine
reported by others (Breves and Schroder, 1991; Ray et
al., 2012b).
The lack of any effect of phytase supplementation
on P flow, apparent P digestibility, and retained P was
due, at least in part, to the use of late-lactation cows
fed dietary P in excess of requirements. Although the
current experimental model has limitations in terms of
measuring effects on digestion and retention, it is still
useful for testing the effect of phytase supplementa-
tion on P bioavailability via measurement of ileal Pi
flow and for estimating capacity for P absorption in
the large intestine. Existing models on P digestion rely
heavily on small ruminant data (Vitti et al., 2000; Dias
et al., 2006) and very limited lactating cow data (Hill
et al., 2008). Data in the present study may be used to
improve published models of P digestion, absorption,
and metabolism.
CONCLUSIONS
Phytase acted rapidly to degrade Pp to Pi in the
TMR; subsequent effects on P flows through the di-
gestive tract were modest. The tendency of reduced
DM digestibility observed with finely chopped forage
affected flow and absorption of P, increasing escape of
P and Pi from the small intestine and increasing fecal P
excretion. Net absorption of up to 25% of ileal P from
the large intestine was observed.
ACKNOWLEDGMENTS
This project was supported by National Research
Initiative Competitive Grant no. 2009-55206-05267
from the USDA Cooperative State Research, Educa-
tion, and Extension Service (Washington, DC), and
by DSM Nutrition (Parsippany, NJ). Authors Jarrett
and Ray received fellowship support from the John Lee
Pratt Foundation.
REFERENCES
AOAC. 1984. Official Methods of Analysis. 14th ed. AOAC, Wash-
ington, DC.
Beauchemin, K. A., W. Z. Yang, and L. M. Rode. 2003. Effects of par-
ticle size of alfalfa-based dairy cow diets on chewing activity, rumi-
nal fermentation, and milk production. J. Dairy Sci. 86:630–643.
Block, H. C., G. E. Erickson, and T. J. Klopfenstein. 2004. Review:
Re-evaluation of phosphorus requirements and phosphorus reten-
tion of feedlot cattle. Prof. Anim. Sci. 20:319–329.
Bohlke, R. A., R. C. Thaler, and H. H. Stein. 2005. Calcium, phospho-
rus, and amino acid digestibility in low-phytate corn, normal corn,
and soybean meal by growing pigs. J. Anim. Sci. 83:2396–2403.
Bravo, D., F. Meschy, C. Bogaert, and D. Sauvant. 2000. Ruminal
phosphorus availability from several feedstuffs measured by the
nylon bag technique. Reprod. Nutr. Dev. 40:149–162.
Breves, G., and B. Schroder. 1991. Comparative aspects of gastrointes-
tinal phosphorus metabolism. Nutr. Res. Rev. 4:125–140.
Clark, W. D. J., J. E. Wohlt, R. L. Gilbreath, and P. K. Zajac. 1986.
Phytate phosphorous intake and disappearance in the gastrointes-
tinal tract of high producing dairy cows. J. Dairy Sci. 69:3151–
3155.
Cohen, R. D. H. 1980. Phosphorus in rangeland ruminant nutrition: A
review. Livest. Prod. Sci. 7:25–37.
Dias, R. S., E. Kebreab, D. M. S. S. Vitti, A. P. Roque, I. C. S. Bueno,
and J. France. 2006. A revised model for studying phosphorus and
calcium kinetics in growing sheep. J. Anim. Sci. 84:2787–2794.
Fellner, V., M. F. Weiss, A. T. Belo, R. L. Belyea, F. A. Martz, and
A. H. Orma. 1988. Urine cup for collection of urine from cows. J.
Dairy Sci. 71:2250–2255.
Forar, F. L., R. L. Kincaid, R. L. Preston, and J. K. Hillers. 1982.
Variation of inorganic phosphorus in blood plasma and milk of
lactating cows. J. Dairy Sci. 65:760–763.
France, J., and R. C. Siddons. 1986. Determination of digesta flow by
continuous marker infusion. J. Theor. Biol. 121:105–119.
Godoy, S., and F. Meschy. 2001. Utilisation of phytate phosphorus by
rumen bacteria in a semi-continuous culture system (Rusitec) in

8. 418 JARRETT ET AL.
Journal of Dairy Science Vol. 97 No. 1, 2014
lactating goats fed on different forage to concentrate ratios. Re-
prod. Nutr. Dev. 41:259–265.
Harvatine, D. I., J. E. Winkler, M. Devant-Guille, J. L. Firkins, N. R.
St-Pierre, B. S. Oldick, and M. L. Eastridge. 2002. Whole linted
cottonseed as a forage substitute: Fiber effectiveness and digestion
kinetics. J. Dairy Sci. 85:1988–1999.
Hill, S. R., K. F. Knowlton, E. Kebreab, J. France, and M. D. Hani-
gan. 2008. A model of phosphorus digestion and metabolism in the
lactating dairy cow. J. Dairy Sci. 91:2021–2032.
Huhtanen, P., P. G. Brotz, and L. D. Satter. 1997. Omasal sampling
technique for assessing fermentative digestion in the forestomach
of dairy cows. J. Anim. Sci. 75:1380–1392.
Kammes, K. L., and M. S. Allen. 2012. Nutrient demand interacts
with grass particle length to affect digestion responses and chew-
ing activity in dairy cows. J. Dairy Sci. 95:807–823.
Kemme, P. A., A. W. Jongbloed, Z. Mroz, J. Kogut, and A. C.
Beynen. 1999. Digestibility of nutrients in growing–finishing pigs
is affected by Aspergillus niger phytase, phytate and lactic acid
levels: 1. Apparent ileal digestibility of amino acids. Livest. Prod.
Sci. 58:107–117.
Kincaid, R. L., D. K. Garikipati, T. D. Nennich, and J. H. Harrison.
2005. Effect of grain source and exogenous phytase on phosphorus
digestibility in dairy cows. J. Dairy Sci. 88:2893–2902.
Knowlton, K. F., and J. H. Herbein. 2002. Phosphorus partitioning
during early lactation in dairy cows fed diets varying in phospho-
rus content. J. Dairy Sci. 85:1227–1236.
Knowlton, K. F., M. L. McGilliard, Z. Zhao, K. G. Hall, W. Mims, and
M. D. Hanigan. 2010. Effective nitrogen preservation during urine
collection from Holstein heifers fed diets with high or low protein
content. J. Dairy Sci. 93:323–329.
Knowlton, K. F., C. M. Parson, C. W. Cobb, and K. F. Wilson. 2005.
Exogenous phytase plus cellulase and phosphorus excretion in
dairy cows. Prof. Anim. Sci. 21:212–216.
Knowlton, K. F., M. S. Taylor, S. R. Hill, C. Cobb, and K. F. Wilson.
2007. Manure nutrient excretion by lactating cows fed exogenous
phytase and cellulase. J. Dairy Sci. 90:4356–4360.
Kononoff, P. J., A. J. Heinrichs, and D. R. Buckmaster. 2003. Modi-
fication of the Penn State forage and total mixed ration particle
separator and the effects of moisture content on its measurements.
J. Dairy Sci. 86:1858–1863.
Krause, K. M., D. K. Combs, and K. A. Beauchemin. 2002. Effects of
forage particle size and grain fermentability in mid lactation cows.
I. Milk production and diet digestibility. J. Dairy Sci. 85:1936–
1946.
Liao, S. F., A. K. Kies, W. C. Sauer, Y. C. Zhang, M. Cervantes, and
J. M. He. 2005. Effect of phytase supplementation to a low- and
a high-phytate diet for growing pigs on the digestibilities of crude
protein, amino acids, and energy. J. Anim. Sci. 83:2130–2136.
Matsui, T., Y. Murakami, H. Yano, H. Fujikawa, T. Osawa, and Y.
Asai. 1999. Phytate and phosphorus movements in the digestive
tract of horses. Equine Vet. J. Suppl. 30:505–507.
Mertens, D. R. 1997. Creating a system for meeting the fiber require-
ments of dairy cows. J. Dairy Sci. 80:1463–1481.
Mertens, D. R. 2002. Gravimetric determination of amylase-treated
neutral detergent fiber in feeds using refluxing in beakers or cru-
cibles: Collaborative study. J. AOAC Int. 85:1217–1240.
Morse, D., H. Head, C. J. Wilcox, H. H. Van Horn, C. D. Hissem, and
B. Harris Jr. 1992. Effects of concentration of dietary phosphorous
on amount and route of excretion. J. Dairy Sci. 75:3039–3049.
Murphy, J., and J. P. Riley. 1958. A single-solution method for de-
termination of soluble phosphate in natural waters. J. Mar. Biol.
Assoc. U. K. 37:9–14.
NRC. 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl.
Acad. Press, Washington, DC.
Nelson, T. S., L. W. Ferrara, and N. L. Storer. 1968. Phytate phos-
phorus content of feed ingredients derived from plants. Poult. Sci.
47:1372–1374.
Park, W. Y., T. Matsui, C. Konishi, S. W. Kim, F. Yano, and H.
Yano. 1999. Formaldehyde treatment suppresses ruminal degrada-
tion of phytate in soyabean meal and rapeseed meal. Br. J. Nutr.
81:467–471.
Park, W. Y., T. Matsui, F. Yano, and H. Yano. 2000. Heat treatment
of rapeseed meal increases phytate flow in the duodenum of sheep.
Anim. Feed Sci. Technol. 88:31–37.
Park, W. Y., T. Matsui, and H. Yano. 2002. Post-ruminal phytate
degradation in sheep. Anim. Feed Sci. Technol. 101:55–60.
Poulsen, H. D., K. Blaabjerg, J. V. Norgaard, and M. A. Ton Nu.
2012. High-moisture air-tight storage of barley and wheat im-
proves nutrient digestibility. J. Anim. Sci. 90(Suppl. 4):242–244.
Ravindran, V., P. H. Selle, G. Ravindran, P. C. H. Morel, A. K. Kies,
and W. L. Bryden. 2001. Microbial phytase improves performance,
apparent metabolizable energy, and ileal amino acid digestibility of
broilers fed a lysine-deficient diet. Poult. Sci. 80:338–344.
Ray, P. P., J. Jarrett, and K. F. Knowlton. 2013. Effect of dietary
phytate on phosphorus digestibility in dairy cows. J. Dairy Sci.
96:1156–1163.
Ray, P. P., C. Shang, R. O. Maguire, and K. F. Knowlton. 2012a.
Quantifying phytate in dairy digesta and feces: Alkaline extrac-
tion and high performance ion chromatography. J. Dairy Sci.
95:3248–3258.
Ray, P. P., C. Shang, R. E. Pearson, and K. F. Knowlton. 2012b.
Disappearance of infused phytate from the large intestine of dairy
heifers. J. Dairy Sci. 95:5927–5935.
Reynal, S. M., and G. A. Broderick. 2005. Effect of dietary level of
rumen-degradable protein on production and nitrogen metabolism
in lactating dairy cows. J. Dairy Sci. 88:4045–4064.
Rode, L. M., D. C. Weakley, and L. D. Satter. 1985. Effect of forage
amount and particle size in diets of lactating dairy cows on site
of digestion and microbial protein synthesis. Can. J. Anim. Sci.
65:101–111.
Rounds, M. A., and S. S. Nielsen. 1993. Anion-exchange high perfor-
mance liquid chromatography with post-column detection for the
analysis of phytic acid and other inositol phosphates. J. Chro-
matogr. A 653:148–152.
Udén, P., P. E. Colucci, and P. J. Van Soest. 1980. Investigation of
chromium, cerium, and cobalt as markers in digesta. J. Sci. Food
Agric. 31:625–632.
Valk, H., J. A. Metcalf, and P. J. A. Withers. 2000. Prospects for
minimizing P excretion in ruminants by dietary manipulation. J.
Environ. Qual. 29:28–36.
Valk, H., L. B. J. Sebek, and A. C. Beynen. 2002. Influence of phos-
phorus intake on excretion and blood plasma and saliva concentra-
tions of phosphorus in dairy cows. J. Dairy Sci. 85:2642–2649.
Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for
dietary fiber, neutral detergent fiber, and nonstarch polysaccha-
rides in relation to animal nutrition. J. Dairy Sci. 74:3583–3597.
Vitti, D. M., E. Kebreab, J. B. Lopes, A. L. Abdalla, F. F. De Car-
valho, K. T. De Resende, L. A. Crompton, and J. France. 2000.
A kinetic model of phosphorus metabolism in growing goats. J.
Anim. Sci. 78:2706–2712.
Voelker, J. A., G. M. Burato, and M. S. Allen. 2002. Effects of pretrial
milk yield on responses of feed intake, digestion, and production to
dietary forage concentration. J. Dairy Sci. 85:2650–2661.
Wise, A., and D. J. Gilburt. 1982. Phytate hydrolysis by germfree and
conventional rats. Appl. Environ. Microbiol. 43:753–756.
Wu, Z., L. D. Satter, A. J. Blohowiak, R. H. Stauffacher, and J. H.
Wilson. 2001. Milk production, phosphorus excretion, and bone
characteristics of dairy cows fed different amounts of phosphorus
for two or three years. J. Dairy Sci. 84:1738–1748.
Yang, W. Z., and K. A. Beauchemin. 2007. Altering physically effec-
tive fiber intake through forage proportion and particle length:
Digestion and milk production. J. Dairy Sci. 90:3410–3421.
Yanke, L. J., L. B. Selinger, and K. J. Cheng. 1999. Phytase activity of
Selenomonas ruminantium: A preliminary characterization. Lett.
Appl. Microbiol. 29:20–25.
Zebeli, Q., M. Tafaj, I. Weber, J. Dijkstra, H. Steingass, and W. Dro-
chner. 2007. Effect of varying dietary forage particle size in two
concentrate levels on chewing activity, ruminal mat characteristics,
and passage in dairy cows. J. Dairy Sci. 90:1929–1942.