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).
- Canadian Journal of Animal Science - CAN J ANIM SCI. 01/1989; 69(1):181-186.
- British Journal Of Nutrition 02/1966; 20(4):795-805. · 3.30 Impact Factor
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ABSTRACT: Du‰ková D., R. Dvofiák, V. Rada, J. Doubek, M. Marounek: Concentration of Phytic Acid in Faeces of Calves Fed Starter Diets. Acta Vet. Brno 2001, 70: 381-385. Ruminants utilize phytic acid well. The digestive system of young calves, however, is less developed than that of adult animals. The aim of our study was to decide whether phytic acid hydrolysis in the digestive tract of calves is complete or not. Thus, rectal samples of faeces were collected from 16 crossbred calves fed starter diets. Faeces of calves were sampled at the age of 1, 6 and 13 weeks. In starter concentrates and faeces the phytic acid content was determined by capillary isotachophoresis. Two commercial starters containing 9.17 and 12.80 g of phytic acid per kg of dry matter (37.6 and 50.1% of the total P) were offered ad libitum from the 4th day of life. Calves were weaned from milk at the age of 6 weeks. Phytic acid was determined in 30 out of 37 faecal samples. Its concentration varied widely from 1 mg to 2.34 g per kg of dry matter. In some samples, the quantity of phytic acid was insufficient for a precise determination. Phytic acid phosphorus represented 0 to 8.9% of the total P in faeces. The highest phytic acid concentrations were found in faeces of calves at the age of 6 weeks, just after weaning. On average, faeces of 1, 6 and 13 weeks old calves contained 8 ± 4, 477 ± 265 and 275 ± 211 mg phytic acid per kg DM, respectively (means ± SEM). It can be concluded that a part of ingested phytic acid apparently escapes the hydrolysis in the digestive tract of calves fed commercial starters. Nutrition, concentrated feed, phosphorus
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
Association of Official Analytical Chemists.
1990. Official Methods of Analysis. (15th Ed.).
AOAC, Arlington VA.
Burkholder, K. M., A. D. Guyton, J. M. Mckin-
ney, and K. F. Knowlton. 2004. The effect of
steam flaked or dry ground corn and supple-
mental phytic acid on nitrogen partitioning in
lactating cows and ammonia emission from
manure. J. Dairy Sci. 87:2546.
Clark, W. D. J., J. E. Wohlt, R. L. Gilbreath,
and P. K. Zajac. 1986. Phytate phosphorous in-
take and disappearance in the gastrointestinal
tract of high producing dairy cows. J. Dairy
Dayrell, M. S., and M. Ivan. 1989. True absorp-
tion of phosphorus in sheep fed corn silage
and silage supplemented with dicalcium or
rock phosphate. Can. J. Anim. Sci. 69:181.
Duskova, D., R. Dvorak, V. Rada, J. Doubek,
and M. Marounek. 2001. Concentration of
phytic acid in faeces of calves fed starter diets.
Acta Vet. Brno. 70:381.
Godoy, S., and F. Meschy. 2001. Utilization of
phytate phosphorus by rumen bacteria in a
semi-continuous culture system (Rusitec) in
lactating dairy goats fed on different forage to
concentrate ratios. Reprod. Nutr. Dev. 41:259.
Guyton, A. D., J. M. McKinney, and K. F.
Knowlton. 2003. The effect of steam flaked or
ground corn and supplemental phytic acid on
ruminal phytase activity and P balance in lac-
tating cows. J. Dairy Sci. 86:3972.
Hill, B. E., S. L. Hankins, J. F. Kearney, J. D.
Arseneau, D. T. Kelly, S. S. Donkin, B. T. Rich-
ert, and A. L. Sutton. 2002. Effects of feeding
low phytic acid corn and phytase on phospho-
rus balance in lactating dairy cows. J. Dairy
Sci. 85 (Suppl. 1):44.
Knowlton, K. F., and J. H. Herbein. 2002.
Phosphorus balance during early lactation in
dairy cows fed diets varying in phosphorus
content. J. Dairy Sci. 85:1227.
Knowlton, K. F., J. H. Herbein, M. A. Meister-
Weisbarth, and W. A. Wark. 2001. Nitrogen
and phosphorous partitioning in lactating Hol-
stein cows fed different sources of dietary pro-
tein and phosphorus. J. Dairy Sci. 84:1210.
Knowlton, K. F., J. S. Radcliffe, C. L. Novak,
and D. A. Emmerson. 2004. Animal manage-
ment to reduce phosphorus losses to the envi-
ronment. J. Anim. Sci. 82E:173.
Martz, F. A., A. T. Belo, M. F. Weiss, and R. L.
Belyea. 1990. True absorption of calcium and
phosphorus from alfalfa and corn silage fed to
lactating cows. J. Dairy Sci. 73:1288.
Martz, F. A., A. T. Belo, M. F. Weiss, and R. L.
Belyea. 1999. True absorption of calcium and
phosphorus from corn silage fed to nonlactat-
ing, pregnant dairy cows. J. Dairy Sci. 82:618.
Morse, D., H. H. Head, and C. J. Wilcox.
1992b. Disappearance of phosphorous in phy-
tate from concentrates in vitro from rations
fed to lactating dairy cows. J. Dairy Sci.
Morse, D., H. Head, C. J. Wilcox, H. H. V.
Horn, C. D. Hissem, and B. Harris, Jr. 1992a.
Effects of concentration of dietary phospho-
rous on amount and route of excretion. J.
Dairy Sci. 75:3039.
National Research Council. 2001. Nutrient Re-
quirements of Dairy Cattle. (7th Rev. Ed.).
Natl. Acad. Sci., Washington, DC.
Reid, R. L., and M. C. Franklin. 1947. The utili-
zation of phytate phosphorus by sheep. The
Austr. Vet. J. 25:136.
Van Soest, P. J., J. B. Robertson, and B. A.
Lewis. 1991. Methods for dietary fiber, neutral
detergent fiber, and nonstarch polysaccharides
in relation to animal nutrition. J. Dairy Sci.
Wu, Z., L. D. Satter, A. J. Blohowiak, R. H.
Stauffacher, and J. H. Wilson. 2001. Milk pro-
duction, estimated phosphorus excretion, and
bone characteristics of dairy cows fed different
amounts of phosphorus for two or three
years. J. Dairy Sci. 84:1738.
Wu, Z., L. D. Satter, and R. Sojo. 2000. Milk
production, reproductive performance, and fe-
cal excretion of phosphorus by dairy cows fed
three amounts of phosphorus. J. Dairy Sci.
Yanke, L. J., H. D. Bae, L. B. Selinger, and K. J.
Cheng. 1998. Phytase activity of anaerobic ru-
minal bacteria. Microbiology 144:1565.
Young, V. R., G. P. Lofgreen, and J. R. Luick.
1966. The effects of phosphorus depletion,
and of calcium and phosphorus intake, on
the endogenous excretion of these elements
by sheep. Br. J. Nutr. 20:795.