Effect of Heat Treatment of Alfalfa Prior to Ensiling on Nitrogen
Solubility and In Vitro Ammonia Production
I. B. MANDELL, 1 D. N. MOWAT, 1 W. K. BILANSKI, 2 and S. N. RAI 3
ABSTRACT
Two experiments were conducted to
assess heat ~eatment of alfalfa prior to
ensiling. In Experiment 1, direct-cut al-
falfa (26% DM) was subjected to dry heat
(air temperature 400"C) for either 0, 7,
14, 21, or 29 s prior to ensiling. Wilted
alfalfa (50% DM) was treated similarly
for either 0, 5, or 6 s. Heat treatment
decreased the soluble N content of silage
without inducing heat damage. Across all
hours of incubation, heating prior to ensi-
ling decreased in vitro NH 3 N production
in comparison with untreated silage (di-
rect-cut or wilted). Duration of treatment
affected NH3 production. Treatment of
direct-cut material for at least 14 s was
necessary to decrease NH3 production in
comparison with unheated alfalfa or al-
falfa treated for 7 s prior to ensiling.
Treatment (6 s duration) following wilt-
ing decreased NH3 production in compar-
ison with heat-treated, direct-cut alfalfa
(7, 14, 29 s treatments). In Experiment 2,
direct-cut alfalfa (36% DM) was sub-
jected to microwave heating for either 0,
30, 60, 120, or 180 s prior to ensiling.
Heat treatment decreased the soluble N
content of silage. Treatment for at least
60 s resulted in a further decrease in N
solubility. In addition, treatment for at
least 60 s was necessary to decrease NH 3
production. Thus, short-term heat treat-
ment of alfalfa prior to ensiling may be
an effective alternative for improving the
utilization of silage N in ruminants.
Received September 30. t988.
Accepted February 22, 1989.
1Department of Animal and Poultry Science.
~School of Engineering.
3present address: National Dairy Research Institute,
Kamal 132001, 1-Iaryana,India.
1989 J Dairy Sci 72:2046-2054
University of Guelph
Guelph, Ontario N1G 2Wl, Canada
INTRODUCTION
The CP content of high quality hay crop
silages exceeds the recommended requirements
for young growing calves (18). However, sev-
eral recent studies have shown a marked im-
provement in the performance of calves fed
either early cut, wilted alfalfa-grass silages (16)
or direct-cut grass silages (5, 25) when supple-
mented with a protein source of low rumen
degradability. This paradox is due to the fact
that forage protein undergoes extensive degra-
dation during wilting and the ensilage process
(6, 23). Various chemicals (formic acid, formal-
dehyde, NH3) have been used as additives dur-
ing ensiling to improve silage stability and N
utilization (7, 13). However, these additives do
not always prevent extensive proteolysis during
ensiling (15) nor improve animal performance
(7).
Artificial drying of forages improves the
utilization of forage protein by the ruminant.
Drying decreases N solubility and rumen degra-
dation of forage protein, and increases micro-
bial protein synthesis in the rumen (9, 15).
Overall, this increases the amount of amino
acids entering the small intestine (9, 23). Un-
fortunately, due to costs, artificial drying has
not been developed for on-farm processing, and
few producers are able to take advantage of this
technology. Recently, Charmley and Veira (4)
demonstrated that steam treatment (60 s dura-
tion) of alfalfa prior to ensiling could inhibit
proteolysis and markedly improve the utiliza-
tion of silage N by the ovine. The objective of
this study was to determine the effects of heat
treatment (HT) of alfalfa prior to ensiling on N
solubility and availability to rumen microbes.
MATERIALS AND METHODS
Experiment 1
First-cut alfalfa (early bloom) was cut and
then chopped by a forage harvester when the
2046
 HEAT TREATMENT OF FORAGES PRIOR TO ENSILING
DM content was 26%. Random portions of the
chopped forage were allowed to wilt 24 h until
the DM attained 50%. Direct-cut and wilted
alfalfa were heat treated at 400°C (air ambient
temperature), using a continuous flow unit (28)
in which heat was supplied by 26, 750-W cal-
rod heaters. Forage transit time through the
continuous flow unit was either 0, 7, 14, 21, or
29 s for direct-cut alfalfa and either 0, 5, or 6 s
for wilted alfalfa. Wilted alfalfa could not be
heat treated for more than 6 s due to the
development of charring and combustion. Each
treatment was processed in quadruplicate. Im-
mediately posttreatment, duplicate samples of
alfalfa were either frozen at -20°C or ensiled in
polyethylene containers (3200 ml capacity) for
35 d. Nonensiled and ensiled alfalfa were
freeze-dried and then ground to pass through a
2-mm screen (Christy-Norris Hammermill
Christy and Norris Ltd., Chelmsford, England).
Analyses of DM, organic matter (OM), and
total N (TN) were conducted according to
AOAC (1) procedures. Neutral detergent fiber,
ADF, and ADIN were determined according to
the sequential methods described by Van Soest
and Robertson (24). Hot water insoluble N was
determined according to Goering and Van
Soest (8) and soluble N calculated. Experimen-
tal design was a factorial arrangement within a
randomized complete block with the two silos
(replicates) serving as blocks. Analysis of vari-
ance was conducted on the chemical composi-
tion data with storage form of alfalfa, HT, and
blocks as factors in the model. Differences
among treatment means were determined by
nonorthogonal contrasts (22).
Samples were selected from wilted and di-
rect-cut alfalfa (nonensiled) and silage and then
incubated at 39"C with 10 ml of rumen buffer
(8) and 5 ml of strained rumen fluid for 0, 1, 3,
6, 12, and 24 h. Dextrose (.92 g/L) was added
to the rumen buffer mixture to provide addi-
tional energy for rurnen microorganisms (8).
Blank incubations without substrate were con-
ducted at all hours of incubation to determine
NH 3 production from rumen fluid alone. Prior
to the a.m. feeding, rumen fluid was obtained
from a nonlactating, rumen-fistulated dairy cow
fed an alfalfa/grass hay. Rumen fluid was
strained through four layers of cheesecloth and
then kept at 39°C prior to use. After dispensing
2047
5 ml of rumen buffer into each incubation tube,
10 ml of the complete incubation mixture (5 ml
rumen fluid, 5 ml rumen buffer) were added
prior to the tubes being gassed with CO2, stop-
pered with bunsen valves, and then incubated in
a water bath at 39"C.
Incubations were stopped at the appropriate
intervals by the addition of 2 ml .4 N H2SO4.
Samples of tube contents were collected after
vortexing and then centrifuged at 13,000 x g
for 3 rain. Supernatant was kept frozen at
-20"C until analyzed for NH 3. Ammonia N
concentrations were determined using a phenol-
hypochlorite colorimetric procedure (2). Accu-
mulation of NH3 N at each interval was calcu-
lated as the difference between NH 3 N concen-
trations of tubes containing substrate and NH 3
N concentrations in blank incubations.
Experimental design for the NH3 N data was
a randomized complete block with the two runs
serving as blocks. For each hour of incubation,
analysis of variance was conducted with HT
and blocks as factors in the model. Differences
among treatment means were determined by
nonorthogonal contrasts (22).
Experiment 2
Third-cut alfalfa (early bloom) was cut and
then chopped by a forage harvester when the
DM was 36%. Fiberboard trays containing 100
g fresh weight of alfalfa were placed in a
conventional kitchen microwave (MW) oven
(700 W, 2450 MHz) and then heat treated for
either 0, 30, 60, 120, or 180 s. Each HT was
replicated 20 times. After treatment, duplicate
samples of alfalfa were ensiled in polyethylene
containers (550 ml capacity) for 35 d. A sample
of nonensiled, nonheat-treated alfalfa was fro-
zen at-20"C. After ensiling, sample processing,
chemical analyses, and in vitro NH3 production
were conducted according to the methods de-
scribed in Experiment 1. A randomized com-
plete block design was used in which the two
silos (replicates) served as blocks. Analysis of
variance was conducted on the chemical com-
position and in vitro NH 3 data with HT and
blocks serving as factors in the model. Differ-
ences among treatment means were determined
by nonorthogonal contrasts.
Journal of Dairy Science Vol. 72, No. 8, 1989
2048 MANDELL ET AL.

RESULTS AND DISCUSSION

Experiment 1
Effect of HT with the continuous flow sys-
O tem on the chemical composition of alfalfa is in
Table 1. Heat treatment increased (P<.01) the
DM content of alfalfa (direct-cut or wilted).
The loss in moisture may be due in part to
.
syneresis associated with thermal denaturation
~ ~ .
N~ of soluble proteins from the disruption of hy-
drogen bonding (20). Increasing the duration of
..--,k HT further increased (P<.01) DM content of
direct-cut alfalfa. Heat treating direct-cut alfalfa
. . . . . . .y,~
could be advantageous to producers by increas-
ing forage DM prior to ensiling. This could
~ v ' ~ prevent nulrient losses during wilting without
.9.0 the potential problems of effluent run-off from
=o ensiling a low DM forage (26). Heat treating
wilted alfalfa may present a storage problem
due to the resultant high DM and the potential
for heat damage (10).
Heat treatment increased (P<.01) the NDF
-' e.,
content of wilted alfalfa but did not affect the
NDF content of direct-cut alfalfa. Although
artificial heating has been shown to increase the
NDF and ADF content of alfalfa haylage (10),
8 the ADF content of alfalfa was unaffected by
HT in our study. The TN content of alfalfa was
R~ ~ ~ unaffected (P>.10) by HT and is in agreement
with Yu and Veira (29). Heat treatment de-
creased (P<.01) N solubility (or increased the
t-
C~ HWIN content) in alfalfa. McDonald (15) noted
that artificial drying of feedstuffs can decrease
t"q OO ~ ,.,.., N solubility due to the formation of new link-
ages within and between peptide chains. These
O
.,~ ~ newly formed linkages may be resistant to pro-
~N tease activity or prevent enzymatic access to
adjacent peptide bonds. In alfalfa, 32 to 39% of
the total leaf protein is composed of a single
soluble protein called Fraction-I or ribulose 1,5-
biphosphate carboxylase (14). Although this
" ~ ~r ~.~ protein is totally denatured at a treatment tem-
perature of 80°C for 5 min, partial denaturation
will occur at temperatures as low as 50°C (20).
Wilting alone did not affect (P>.10) N solu-
bility. This is in agreement with McDonald
z .~ ~
(15), who stated that wilting prior to ensiling
m does not necessarily inhibit proteolysis nor pro-
vide any beneficial effect in preventing protein
al
breakdown during ensiling. In contrast, Janicki
and Stallings (10) reported that as the DM
[-, g

Journal of Dairy Science Vol. 72, No, 8, 1989

 HEAT TREATMENTOF FORAGES PRIOR TO ENSILING 2049

content of forage to be ensiled decreased, N

solubility after ensiling increased. Increasing
the duration of HT decreased (P<.05) N solu-
bility. In the present study, HT of direct-cut
alfalfa for 21 and 29 s prevented a decrease in
N solubility and may provide an alternative to
wilting alfalfa prior to ensiling. Heat treatment
for short times (7 and 14 s, direct-cut alfalfa; 5
s, wilted alfalfa) may not inactivate plant en-
zymes totally (14, 21), enabling moderate pro-
teolysis.
Excessive heating of forages increases risk
of heat damage, thus limiting N availability in
the forage (3, 12). Although HT increased
(P<.05) the ADIN content of alfalfa, all values
in our study were lower than the 10% figure
suggested by Janicki and Stallings (10) to rep-
resent limited heat damage.
Total N and soluble N content of alfalfa
differed (P<.05) between storage forms (nonen-
siled vs. ensiled). Although the difference in
_= TN content (nonensiled, 3%; ensiled 3.05%)
was small, the solubility of ensiled alfalfa ex-
e-
ceeded (P<.01) that of nonensiled alfalfa (non-
ensiled, 36.4% of TN; ensiled, 45.3% of TN).
c-
The increase in N solubility upon ensiling is
O attributed to plant proteolysis during the ensi-
ling process (15).
A storage form by treatment interaction
(P<.01) occurred for N solubility (Table 2).
O The interaction was due to a decrease in N
solubility from ensiling of control (direct-cut
O
._= and wilted) alfalfa and alfalfa heat treated for
either 5, 7, or 14 s. In contrast, ensiling did not
affect N solubility in alfalfa heat treated for 21
=o or 29 s (direct-cut alfalfa) or 6 s (wilted alfal-
fa). According to Charmley and Veira (4), HT
reduces proteolysis during the ensilage process,
oJ
resulting in higher concentrations of insoluble
N and lower concentrations of NH 3 N. The
"o

E
difference in N solubility between control and
heat-processed silages in the present study are
~o quite similar to those previously reported (4).
In the latter work, HT prior to ensiling in-
()
creased the flow of non-NH 3 N to the duode-
num in sheep.
Effect of HT on NH 3 N production in vitro
t~ z is in Table 3. Although rumen fluid was
c-i obtained from the same fistulated cow on both
days of incubation, run-to-run variation (P<.01)
.l
occurred throughout all hours of incubation.
b- This resulted in large SE. Broderick (2) also

Journal of Dairy Science Vol. 72, No. 8, 1989

 tO

g
<
o T A B L E 3. Effect o f heat treatment (continuous flow system) prior to ensiling on in vitro N H 3 production (mg NH3-N/g N incubated) 1 from alfalfa.

Duration o f heat treatment

Z Direct-cut alfalfa Wilted alfalfa,
P
Nonensiled Ensiled ensiled Contrast 2
Incubation 0 s 0 s 7 s 14 s 29 s 0 s 6 s SE 1 2 3 4 5 6

(h)
0 10.3 74.0 75.7 42.4 45.7 54.5 19.2 35.5 ** * ? ** *
1 23.5 89.4 85.9 51.8 46.4 59.4 34.8 46.7 ** * 1" , *
3 33.4 90.9 97.2 66.6 51.6 60.7 32.6 38.7 * t t , ** ,
6 59.8 122.6 112.7 67.6 70.3 73.7 22.7 71.9 * * ** * r-
12 83.9 182.7 125.3 40.9 30.1 113.2 9.9 86.0 ** ** * ** ** ,-]
24 203.1 255.0 200.8 ! 17.0 126.8 226.6 88.9 42.3 * ** ** * ** **

1Ammonia production values are blank-corrected.

2Contrast 1, noncnsiled vs. ensiled, unheated (0 s); Contrast 2 (wilted alfalfa), unheated (0 s) vs. heat treated (6 s); Contrast 3 (direct-cut alfalfa), unheated (0 s) vs. heat
treated (7, 14, 29 s); Contrast 4 (direct-cut alfalfa), 7, 14 s heat treated vs. 29 s heat treated; Contrast 5, direct-cut, heat treated (7, 14, 29 s) vs. wilted, heat treated (6 s); Contrast
6 (direct-cut), 7 s heat treated vs. 14 s heat treated.
*P<.05.
**P<.01.
l p < . 10.
 HEAT TREATMENTOF FORAGES PRIOR TO ENSILING
noted run-to-run variation in NH 3 N production
in vitro using casein as a substrate. In the
present study, run-to-run variation was expect-
ed, as NH3 N concentrations produced from
rumen fluid incubated without substrate dif-
fered between days of incubation. Although all
NH3 N values were corrected for NH 3 N pro-
duction from rumen fluid alone, day-to-day
variation may be present for the degradative
activity of rumen fluid (2). The large run-to-run
variation in NH3 N production at 0 h cannot be
explained as the fermentation is stopped imme-
diately with H2SO4 after addition of the buffer-
rumen fluid inoculum.
With the exception of 6- and 12-h incuba-
tions, Nil 3 N production from the nonensiled
alfalfa was less (P<.05) than that from unheated
silage (direct-cut or wilted). In vivo studies (6,
9) showed that feeding fresh forages increased
the amount of forage amIno acids entering the
small intestine in comparison to feeding si-
lages. The inefficient utilization of silage N by
the ruminant is due to extensive proteolysis
during the ensilage process and to rapid and
complete degmd_ation of NPN in the rumen
(23). Extensive proteolysis occurred in the con-
trol silages as noted in Table 2 by their high
soluble N content. Thus, across all hours of
incubation, HT prior to ensiling decreased
(P<.10) NH 3 N production in both direct-cut
and wilted silages. Heat treatment probably de-
creased N availability to plant and microbial
enzymes due to enzyme inactivation (11) and to
thermal denaturation of protein, which pre-
vented proteolysis during ensiling and in vitro
incubation. This is supported by the similar
concentrations of soluble N in heat-treated si-
lages relative to nonensiled control alfalfa (Ta-
ble 2). Similarly, Krause and Klopfenstein (12)
demonstrated that artificial drying of alfalfa
decreased NH 3 N production in vitro.
Duration of HT affected (P<.05) NH3 N
production in vitro. Across all hours of incuba-
tion, NH3 N production from alfalfa heat-
treated for only 7 s was similar to that from the
control direct-cut silage. However, HT for 14 s
decreased (P<.01) NH 3 N production in com-
parison with HT for 7 s duration. Wilting prior
to HT decreased (P<.05) NH 3 N production in
comparison with direct-cut forage. Wilting de-
2051
creased the time required for HT to reduce N
solubility of silage and to decrease N availabil-
ity to rumen microorganisms.
Experiment 2
Effect of MW processing on the DM content
and N fractions of alfalfa is in Table 4. Micro-
wave heating increased (P<.01) the DM content
of alfalfa. This is consistent with use of the
MW for rapid drying of herbage samples (27).
Increasing duration of MW treatment resulted
in a further increase (P<.01) in DM content. In
addition to increasing forage DM, N solubility
was markedly decreased (P<.01) over that of
control silage. Although MW processing for 30
s decreased N solubility, further decreases in N
solubility occurred by increasing the duration
of MW treatment. Decreases (P<.01) in N solu-
bility by MW treatment is either due to enzyme
inactivation (21) or to thermal denaturation of
protein (11). Lower response in N solubility by
MW treatmem for 30 s may be related to the
sample size (100 g fresh weight) processed in
this study or to the power of the MW system
employed (19). Wolf and Carson (27) reported
that respiration was completely inactivated after
25 g fresh alfalfa was MW processed for 30 s.
In addition, Smith (21) noted that rapid drying
cannot occur if a thick or massive layer of
forage is subjected to heat or freeze drying.
Increasing the duration of MW processing be-
yond 60 s did not result in further decreases
(P>.10) in N solubility. This is probably attrib-
uted to the fact that 70 to 80% of the TN in
fresh alfalfa is true protein (17). Despite in-
creases (P<.05) in the ADIN content of alfalfa
with certain MW treatments, values do not
indicate even moderate heat damage.
Effect of MW treatment on in vitro produc-
tion of NH 3 N is in Table 5. Run-to-run varia-
tion (P<.01) resulted in large SE. Ensiling
increased (P<.01) NH 3 N production at 1 h of
incubation and decreased (P<.05) NH 3 N pro-
duction overall at 12 and 24 h of incubation
compared with nonensiled control forage. This
effect was due to HT prior to ensiling as MW
treatment decreased (P<.05) NH3 N production
at 1, 3, 12, and 24 h of incubation in compari-
son with the control silage. With the exception
of the 12-h incubations, HT for 30 s prior to
ensiling did not affect (P>.10) NH 3 N produc-
Journal of Dairy Science Vol. 72, No. 8, 1989
 ~' TABLE 4. Effect of heat treatment (microwave oven) prior to ensiling on the chemical composition of alfalfa, to

p_.. Duration of heat treatment

Nonensiled Ensiled Contrast 1
I~'. Component 0 s 0 s 30 s 60 s 120 s 180 s SE 1 2 3 4 5
,.7
DM, % 32.6 33.0 30.7 36.5 44.1 52.1 .05 ** ** ** ** **
~. Soluble N, % TN 30.5 57.3 46.8 30.9 24.7 25.9 1.29 * ** **
ADIN, % TN 3.81 5.82 5.17 4.18 5.91 6.92 .14 * *

1Contrast 1, nonensiled, unheated (0 s) vs. ensiled (0, 30, 60, 120, 180 s heat treated); Contrast 2, ensiled, unheated (0 s) vs. ensiled, heat treated (30, 60, 120, 180 s);
Contrast 3, ensiled, unheated (0 s) vs. 30 s heat treated; Contrast 4, 120 s heat treated vs. 180 s heat treated; Contrast 5, 60, 120 s heat treated vs. 180 s heat treated.
*P<.05.
Z **P<.01.
9

01
TABLE 5. Effect of heat treatment (microwave oven) prior to ensiling on in vitro ammonia production (mg NH 3 N/g N incubated) from alfalfa. -1

Duration of heat treatment

Nonensiled Ensiled Contrast 2
Incubation 0 s 0 s 30 s 60 s 120 s 180 s SE 1 2 3 4 5
(h)
0 11.6 48.2 51.3 23.1 21.3 16.6 62.9
1 21.5 64.5 54.1 40.8 27.2 27.9 7.8
3 41.6 69.1 68.2 38. I 33.2 33.0 28.2
6 58.7 87.5 109.1 35.1 12.6 15.7 183.9
12 127.4 185.3 150.9 35.3 t9.0 I 1.7 41.5
24 290.2 300.0 284.3 174.2 158.9 161.6 377.6
1Ammonia production values are blank-corrected.
2Contrast 1, nonensilcd, unheated (0 s) vs. ensiled (0, 30, 60, 120, 180 s heat treatcd); Contrast 2, ensiled, unheated (0 s) vs. ensiled, heat treated (30, 60, 120, 180 s);
Contrast 3, ensiled, unheated (0 s) vs. 30 s heat treated; Contrast 4, 120 s heat treated vs. 180 s heat treated; Contrast 5, 60, 120 s heat treated vs. 180 s heat treated.
*P<.05.
**P<.OI.
 HEAT TREATMENT OF FORAGES PRIOR TO ENSILING
tion in comparison with the control silage.
Longer durations of MW treatment appear to be
required to inactivate plant enzymes or dena-
ture plant protein. Microwave treatment at or
exceeding 60 s decreases in vitro NH 3 N pro-
duction and N solubility from that of the 30 s
MW treatment. Unfortunately, operating costs
associated with long treatment durations would
probably be impractical for on-farm processing
(19). Increasing MW treatment beyond 60 s did
not reduce further (P>.10) NH 3 N production.
Nitrogen solubility was also similar among the
60, 120, and 180 s HT.
CONCLUSIONS
These results suggest that short-term HT of
alfalfa prior to ensiling may be an effective
alternative for improving the utilization of si-
lage N in ruminants. Heat treatment prior to
ensiling decreased N solubility in silage and the
availability of silage N for rumen degradation.
This may increase the amount of rumen micro-
bial protein synthesis as well as forage protein
entering the small intestine. Similar findings
with dehydrated alfalfa have been demonstrated
in vivo in sheep (9). Because the ADIN content
of the silage was only marginally increased by
HT, postruminal digestion of any bypass pro-
tein should not be reduced.
This study was restricted to the effects of
different I-IT on N quality of alfalfa silage.
Other parameters of silage quality (e.g., pH,
lactic acid), while important, were not conduct-
ed. Viability of HT prior to ensiling will be
dependent on installation and operating costs
for the treatment technology, and the resultant
animal performance relative to that using con-
ventional protein supplements.
ACKNOWLEDGMENTS
Financial assistance was provided by the
Ontario Ministry of Agriculture and Food.
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