EFFECT OF ORGANIC ACIDS AND
NON-PROTEIN-NITROGEN 0N
FUNGAL GROWTH, NUTRITIVE VALUE,
FERMENTATEON, AND REFERMENTATSQN
OF CORR SiLAGE AND
HEGH MOiSTURE CORN

Bissertation for the Degree of Ph. D.
MICHéGAN STATE UNWERSITY
DANNY GELBERT BRITT
1973

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EFFECT OF ORGANIC ACIDS AND NON-PROTEIN-NITROGEN 0N

FUNGAL GROWTH, NUTRITIVE VALUE, FERMENTATION, AND
REFERMENTATION 0F CORN SILAGE AND
HIGH MOISTURE CORN

presented by

Danny Gilbert Britt

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in Dairy Science

 

 

and Institute of Nutrition

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ABSTRACT
EFFECT OF ORGANIC ACIDS AND NON—PROTEIN-NITROGEN ON
FUNGAL GROWTH, NUTRITIVE VALUE, FERMENTATION AND
REFERMENTATION 0F CORN SILAGE AND
HIGH MOISTURE CORN
By

Danny Gilbert Britt

Experiments were conducted with corn silage and high moisture
shelled corn to evaluate the preservative value of organic acids and
non-protein-nitrogen additives on the fungal growth during fermentation
and refermentation.

Whole chopped corn (35% BM) in lots of 56 kg was treated with
either propionic, formic, propionic plus formic or propionic plus acetic
acids at 0, 0.5, l or 2%; and with either urea, aqua-ammonia or ammonia-
molasses solution at 0, .2, .4, or .8% added nitrogen. Treated material
was placed in polyethylene bags inside 200 2 drums, evacuated, and
sealed. Forages were sampled and temperatures measured at various in-
tervals during fermentation. After fermentation was completed, 12 kg
portions of the preserved forages were placed in open containers and

stored at 25 C. At various dates during refermentation forages were

D

I'T"

Danny Gilbert Britt

sampled, aerated, and temperatures measured. Samples were analyzed for
VFA, lactic acid, pH, and number and type of fungi.

During refermentation acid treatment at 2% reduced the average
temperatures when compared to controls (19.8° vs 24.4°; P < .01); and
.5 and 2% acid reduced (P < .Ol) the maximum temperature compared to
controls (33.6°, 29.6°, and 37.5°, respectively). Propionic at .5 and
l% was more effective than other acids. Lactic acid production was
significantly depressed by all acids at 2% addition. All treatments
containing propionic acid required more days (P < .01) before visual
mold was detected (7.0, l3.4, 20.0, and 20.5 days; for formic, propi—
onic plus formic, propionic plus acetic and propionic, respectively).
Days until complete molding was also increased by acid treatment. All
acids significantly decreased fungal colonies within 2 days after addi-
tion. During refermentation all treatments at 1% acid exhibited a
rapid increase in number of colonies; however, propionic acid treated
silages showed less fungal growth than those treated with formic acid.

The relative proportion of yeasts was greatest at initiation
of fermentation and decreased at day 40 (from 70 to 10%). During re-
fermentation, growth of yeast again accelerated. The proportion of
Geotrichum was highest at day 40 of fermentation. Aspergillus was
significantly higher at day 40 of fermentation and day 36 of refermen-
tation. No significant amounts of Penicillium were detected at any date.

2

Danny Gilbert Britt

Initial pH increased with nitrogen addition. At .8% nitrogen,
pH values for urea, aqua-anmonia and ammonia-solution were 6.35, 9.88,
and 9.65, respectively. Added nitrogen also resulted in large pH in—
creases during refermentation. Ammonia added at .2% nitrogen increased,
but higher levels depressed lactate production in silages. Upon spoil-
age lactic acid disappeared. Silages treated with the ammonia—solution
required the longest to spoil (P < .01). Ammonia nitrogen reduced total
fungal colony counts (P < .01) at 30 minutes after treatment; but during
fermentation, no differences between treatments were noted. At both .4
and .8% nitrogen, fungal counts were lower (P < .01) than at 0 and 2%.
Treatment did not significantly change the relative proportions of fungi
during spoilage. The majority of fungi were yeast and Geotrichum while
some Aspergillus and Penicillium were identified. However, yeast de-
creased and Geotrichum increased during spoilage on all treatments
(P < .01).

High moisture shelled corn (27% moisture) was treated with:
1) propionic acid (at 1.2%), 2) a mixture of 80% propionic and 20%
acetic acid (1.2%), 3) aqua-ammonia (at .54% NH3) or 4) a commercial
ammonia solution (at .63% NH3). Calculated recovery of nitrogen for
the ammonia treatments after storage was 60% on the ammonia solution
and 36% for the aqua-ammonia while about 80% of the propionic acid was
recovered. No significant changes in nitrogen or acid content were

3

Danny Gilbert Britt

observed during storage. Fungi were reduced by all additives (21 vs 690
colonies/g X 103) 30 minutes after treatment. Fungal colony counts
after 28 days for the aqua-ammonia and control treatments were signifi-
cantly higher than the others. During late storage both anmonia treat-
ments showed increases in fungal colonies. They occurred earlier and
were of greater magnitude for the aqua- than the ammonia—solution.
After 60 days of storage, corn treated with aqua—ammonia heated to 50 C
while the other treatments remained at ambient temperature. After roll-
ing the corn prior to feeding the aqua-ammonia corn had higher fungi
(P < .05) than other treatments and that treated with the ammonia—
solution heated to 30 C. The propionic acid treated corn remained at
ambient temperature (4-10 C) and did not heat after rolling.

Lactating dairy cows (8 per group) readily consumed all corn
in a 5—week feeding trial even though that treated with aqua-ammonia
was heavily molded. No health problems were detected in any of the
cows due to eating the moldy corn. Milk production was 21.2, 18.4, and
19.3 kg for treatments 1, 3, and 4 respectively (P < .109) and persis-

tencies of milk yield (trt/std) averaged 93, 88, and 81% (P < .126).

EFFECT OF ORGANIC ACIDS AND NON-PROTEIN-NITROGEN ON
FUNGAL GROWTH, NUTRITIVE VALUE, FERMENTATION, AND
REFERMENTATION OF CORN SILAGE AND

HIGH MOISTURE CORN

By

Danny Gilbert Britt

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Dairy Science and Institute of Nutrition

1973

“I: DEDICATION
O-

I would like to dedicate this thesis to my father,

 

Mr. Gilbert Britt, and grandfather, Mr. Andrew
Britt, who instilled in the author the desire to

never quit.

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ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation to
Dr. J. T. Huber for his advice and counsel throughout his graduate
program. His support and encouragement have been greatly appreciated.

The author would also like to express his gratitude to other
members of the graduate committee; Dr. H. Hafs, Dr. A. Rogers, and
Dr. R. Luecke for their guidance and counsel during the author's grad-
uate program.

Thanks are extended to Dr. C. A. Lassiter and Dr. J. A. Hoefer
for making the facilities at Michigan State University available for
this research and to Dr. J. w. Thomas for his assistance in obtaining
financial support through NIH.

Appreciation is also extended to Alfred Dutrow, Richard Green-
ing, Judy Ball, and Dr. Roger Neitzel for their help during this re-
search.

The writer wishes to extend his sincere gratitude to his wife,
Carolyn, and son, Danny Joe, for their sacrifices, encouragement, and

assistance during his course of study and manuscript preparation.

iii

TABLE OF CONTENTS

LIST OF TABLES .........................
LIST OF FIGURES .........................
LIST OF APPENDIX TABLES .....................
Section

I. INTRODUCTION .......................
II. REVIEW OF LITERATURE ...................
Fungi Development in Feedstuffs .............

Role of Fungi in Forage and Grain Storage .......

Types of Fungi Present ...............

Role of Fungi in Heating of Feeds ..........

Mycotoxin Production by Fungi During Feed Storage. . .
Conditions under which Toxins are Produced .....
Documented Cases of Mycotoxin Poisoning .......
Toxicity and Secretion in Animals Given a Known

Amount of Mycotoxin ................

Contamination of Human Food Supply Via Fungal Toxin
Consumption of Animals ...............

Methods of Controlling Mycotoxin in Feeds ........

Destruction of Mycotoxins by Treatment ........

Table 0

Section

III.

IV.

f Contents (cont.)

Prevention of Fungal Growth with a Fungicide .....

Acid Treatment of Feeds and Grain ..........
Conclusion ......................
Literature Cited ...................

PART A: FUNGAL GROWTH DURING FERMENTATION AND
REFERMENTATION OF ORGANIC ACID TREATED CORN SILAGE .

Abstract ........................
Introduction ......................
Materials and Methods ..................
Summary and Conclusions .................
Literature Cited ....................
PART B: FUNGAL GROWTH DURING FERMENTATION AND

REFERMENTATION 0F NON—PROTEIN-NITROGEN TREATED

CORN SILAGE .......................
Abstract ........................
Introduction ......................
Materials and Methods ..................
Results and Discussion .................
Summary and Conclusions .................

Literature Cited ....................

Page
16
19
34

36

48
48
49
51
75

76

79
79
8O
81
83
94

95

Table of Contents (cont.)

Page
PART C: PRESERVATION AND ANIMAL PERFORMANCE OF HIGH
MOISTURE CORN TREATED WITH NON-PROTEIN-NITROGEN
AND PROPIONIC ACID ................... 97
Abstract ........................ 97
Introduction ...................... 98
Materials and Methods .................. 100
Results and Discussion ................. 102
Summary and Conclusions ................. 120
Literature Cited .................... 122
SUMMARY AND CONCLUSIONS .................. 124
APPENDIX ......................... 127

vi

LIST OF TABLES

Table Page

1. Effect of Level of Organic Acid Addition on Temperature
Development in Corn Silage During Refermentation. . . . 55

2. Lactic Acid Production (% of BM) in Organic Acid Treated
Corn Silage During Fermentation and Refermentation. . . 57

3. PHs of Organic Acid Treated Corn Silage During
Fermentation and Refermentation ............ 60

4. Acetic Acid Content (% DM) of Organic Acid Treated Corn
Silage During Fermentation and Refermentation ..... 64

5. Propionic Acid Content (% DM) of Organic Acid Treated
Corn Silage During Fermentation and Refermentation. . . 65

6. Correlations of Several Variables During Fermentation
and Refermentation of Organic Acid Treated Corn Silage. 66

7. Number of Days until Fungi Were Noted on Corn Silage,
Complete Spoilage and DM Loss During Fermentation as
Affected by Level and Type of Acid ........... 67

8. Number of Fungal Colonies (per gm X 105) on Different
Media, with or without Novobiocin and/or Rose Bengal. . 68

9. Total Fungal Colonies (total counts/gm x 103) in Organic
Acid Treated Corn Silage During Fermentation and
Refermentation ..................... 71

10. Number of Fungal Colonies Obtained from Organic Acid
Treated Corn Silage as Affected by Date ........ 72

11. Number of Fungal Colonies as Affected by Level of
Acid Addition ..................... 72

List of Tables (cont.)
Table Page

12. Types of Platable Fungal Colonies at Day 1 and 40 During
Fermentation and Day 14 and 36 During Refermentation. . 74

13. Maximum Temperatures, Days until Spoilage and Days until
Maximum Temperature During Refermentation as Affected
by Different Levels and Sources of N Addition to Corn
Silage ......................... 88

14. Total Fungal Colonies (Total Counts/g X 103) in Corn
Silage as Affected by NPN Treatment During
Fermentation and Refermentation ............ 90

15. Relative Proportions of Fungi in Corn Silage Treated
with Various Kinds of NPN ............... 93

16. The pH During Storage of HMC Treated with Organic Acids
or NPN ......................... 104

17. Crude Protein (% of DM) of HMC During Storage Treated
with NPN and Organic Acids ............... 105

18. Propionic Acid (% wet wt) of HMC Treated with NPN and
Organic Acids after Storage .............. 106

19. PH and Protein (% of DM) of HMC Treated with NPN and
Organic Acid During Refermentation ........... 110

20. Propionic Acid (% wet wt) of HMC Treated with NPN and
Organic Acids During Refermentation .......... 111

21. PH and Crude Protein after Rolling of HMC Treated
with NPN and Organic Acids ............... 114

22. Propionic Acid (% wet wt) of HMC after Rolling Treated
with NPN and Organic Acids ............... 115

23. Milk Production Data of Cows Fed HMC Treated with Either
Aqua Ammonia, Propionic Acid, or Ammonia Solution . . . 119

24. Body Weight Gain and Feed Consumption of Cows Fed

Propionic Acid, Aqua Ammonia or Ammonia Solution
Treated HMC ...................... 120

viii

Figure
1.

2.

LIST OF FIGURES

Lactic Acid in Control and 0.5% Acid Treated Corn Silage.
Lactic Acid in Control and 2% Acid Treated Corn Silage. .

Fungal Growth in Corn Silage Treated with 1.0% Acid
from 4 Sources .....................

PH of Corn Silages Treated with .2% N from 3 NPN Sources.
Lactic Acid Concentrations of Corn Silage Treated with
Varying Levels of N from 3 Sources (Averaged from 20

and 86 days of Fermentation) ..............

Lactic Acid in Corn Silage Treated with .2% N from 3 NPN
Sources ........................

Relation of NPN Treatment of Corn Silage to Total Fungal
Colonies During Fermentation and Refermentation . . . .

Fungal Counts of HMC Treated with Ammonia and Organic
Acids During Storage ..................

Relationship of Days of Storage During Refermentation to
Fungal Growth in Propionic and Ammonia Treated HMC. . .

Fungal Colonies After Rolling in Ammonia and Propionic
Treated HMC ......................

Temperature Development After Rolling in HMC Treated
with Ammonia or Propionic Acid .............

Page

59

61

73

84

85

87

91

108

113

117

118

Table

LIST OF APPENDIX TABLES

PH's in NPN Treated Corn Silage During Fermentation and
Refermentation .....................

Lactic Acid Production (% of DM) in NPN Treated Corn
Silage During Fermentation and Refermentation .....

Total Fungal Colonies (per g X 103) of HMC Treated with
Ammonia and Organic Acids ...............

Total Number of Platable Fungal Colonies (X 103 per g)
in HMC Treated with Various Preservatives During
Refermentation .....................

Total Fungal Colonies (per g X 103) in HMC Treated with
Ammonia and Propionic Acid after Rolling ........

Page

127

128

129

130

131

I. INTRODUCTION

Changes in recent years have been oriented toward the storage
of corn silage, haylage and corn, at moistures more susceptible to
fungal growth, especially after aeration upon removal from storage.
These fungal contaminants can produce a wide variety of toxic metabo-
lites called mycotoxins (Lynch, 1972). A survey conducted in the
United States revealed no contamination of the milk supply (Brewington
gt;gl., 1970); however, Purchase and Vooster (1968) reported contamin-
ation of commercially available milk in South Africa.

Organic acids have been reported to be effective in preventing
fungal growth (Sleiman, 1972; Richardson and Halick, 1957). In addi-
tion, Bothast §t_§l, (1973) reported that ammonia may be used as a
fungicide in stored grains. There is the possibility that these
treatments may select for a more potent toxin forming fungi.

The objectives of this thesis were to examine the type and num-
ber of fungi which grew during fermentation and refermentation of non—
protein nitrogen and organic acid treated corn silage and high moisture
corn. The nutritive value of the treated HMC was also compared using

lactating dairy cows.

II. REVIEW OF LITERATURE

Recently emphasis has been placed on treating forages to
decrease losses during storage and prevent spoilage due to fungal
growth. In addition much interest has developed in preserving high
moisture corn (HMC) with volatile fatty acids (VFA).

This review will discuss the problem of mycotoxin formation
in feedstuffs and the effect of chemical treatments on the fungal

growth patterns.

Fungi Development in Feedstuffs

Role of Fungi in Forage and Grain Storage

The problem of fungal contamination of feedstuffs is twofold.
One is the loss of nutrients in feeds due to fungal growth and the
other involves secretion of metabolites which are toxic to animals.
Until recently, most interest has been in the loss of nutrients. Re-
cently, however, due to changes in harvesting methods and more sensi-

tive analytical procedures, concern has developed over the possible

contamination of feeds with toxins, their effects on the animal, and
entrance into the human food supply.

Wogan (1964) stated that ergotism has been known for centuries
and occasional reports have appeared during the past three decades
associating ingestion of mold-contaminated foodstuffs with a variety
of toxicity syndromes in domestic animals and in one instance man.
However there is still a failure to appreciate the significance of

food-borne mycotoxins in problems of animal and human health.

Types of Fungi Present

Different types of fungi are present during harvesting and
storage of grains. These are the field fungi and storage fungi.
Tuite and Christensen (1955) found Alternaria, Cladosporium, and
Fusarium were common in seeds prior to harvest while Aspergillus and
Penicillium appeared after harvest. Christensen (1949) found that
the common field fungi were Alternaria, Helminthosporium, and Fusarium
while Aspergillus and Penicillium were the predominate storage fungi
and grew best at about 30° C. In a survey of fungi in flour, Chris-
tensen and Cohen (1950) found counts ranging from several hundred to
about 5,000 per gram. These counts remained stable for about 2 years
and were predominately Aspergillus and Penicillium. Bothast and co-

workers (1973) reported mold counts of whole corn varied from 102 to

106 in the 1970-1971 crop. Penicillium and Fusarium species predomin-

ated among the molds but Aspergillus, Helminthosporium, Nigrospora,
and Trichoderma were significant.

Not all fungi which grow on grains are toxic. Scott (1965)
isolated 228 strains from domestic cereal and legume crops and of
these, 46 were toxic when grown in pure-culture and fed to Pekin duck-
lings. Christensen and co-workers (1968) isolated 943 fungi strains
in 40 genera from feeds, peanut fruits and seeds of which 54% were

toxic to rats within 7 days. Richard et a1. (1969) isolated 246 fungi

 

from 25 moldy corn samples in Iowa. Extracts of 99 of these isolates
were toxic with the majority of the toxic isolates being Aspergillus
or Penicillium. Burnside gfl;gil. (1957) isolated 13 cultures of mold
from fields where hogs were dying. Two of the 13 cultures produced

toxins. These were identified as Penicillium rubrum and Aspergillus

 

flavus. Semeniuk et a1. (1971) isolated 392 strains of Aspergillus

and found 166 of these were toxigenic.

Role of Fungi in Heating of Feeds

Gilman and Barron (1930) tempered grain to 18% moisture and
sterilized the grain. The grain was then allowed to germinate with
or without fungal inoculation. The presence of fungi imcreased the

temperature over the non-inoculated from 5.2 to 26.4 C depending on

type of grain. They concluded that there was a high probability that
in bins of stored grains the marked increase in temperature may be
ascribed to mold growth. Milner and Geddes (1946) reported that mold
growth was responsible for soybean heating to 50-55 C and was asso-
ciated with the growth of A. glaucus and A, jlgvgs. Milner and Geddes
(1945) found mold proliferation in seed, as visually observed, was
positively correlated with respiratory activity, and with increases

in temperature to 40 C.

In further support Christensen gt_al. (1949) reported a close
correlation between increase in production of C02 and mold population.
Christensen and Gordon (1948) found molds caused the temperature to
rise within a few degrees of the maximum that the molds could endure.
Autoclaved, moist wheat inoculated with 200,000 spares of A, flavgs
per gram heated to 45 C in two days while that inoculated with 0.2
spore per gram took nine days to heat to a comparable temperature.
They concluded that the amount of inoculum originally present has
only a minor effect on eventual heating.

Honig (1969) conducted gas balance tests with silages and
found DM losses increased linearly with the amount of added air.

The digestibility of nutrients as well as the quality and stability
of silages decreased with increased air. Because fungi are aerobic
this air may have been stimulating fungal growth but this was not

mentioned. In support, Federson (1971) found oxidation of high

moisture silages in insulated silos was accompanied by a large rise in
temperature and high DM losses. Temperature increases in the silos
without oxygen present were negligible. He observed that the pH
closely paralleled the added oxygen. Gordon (1968) harvested corn
silage at late maturity (58-63% DM) and early maturity (26-30% DM)

and found digestibility of DM and acid detergent fiber were lower in
the mature silage. Early silage had more VFA and lactic acid and the
late silage had a tendency to heat when fed in hot weather.

Gregory and co-workers (1963) harvested wet hay (60% DM) which
became very hot and contained a large flora of thermophilic fungi.
Actinomycetes and bacteria grew during the first heating with in—
creases in acidity and volatile nitrogen. When fungi grew the pH
rose to 7.0 or above. The wet stack developed a brown acid hay center
containing many spore-forming bacteria but few fungi, surrounded by an
outside layer of moldy hay. According to Thomas and Hillman (1972),
excessive heating during curing of haylage, baled hay or stacked hay
caused carmelization to occur between plant proteins, sugar and water
resulting in a product which was insoluble and indigestible. They
concluded the carmelization effect was small but measurable when
forages heated to 46C, greater at 51.7 C and protein digestibility

was markedly reduced at 57 C. Perry et a1. (1968) suggested that the

 

corn plant may be harvested at a much later stage than hard dough

W- .~ -_-.—

which has been recommended by other workers if stored in gas-tight

silos which would keep fungal contamination to a minimum.

Mycotoxin Production by Fungi During Feed Storage

Conditions under which
Toxins Are Produced

A complete listing of all toxins produced by fungi is too
lengthy for this review, but such a list has been published by Wasser-

man (1968) in which he listed 11 toxins produced by Alternaria, 16 by

Fusarium, 66 by Aspergillus and 98 by Penicillium. Crane et a1.

(1972) suggested four conditions for aflatoxin production. These
were: 1) suitable strain of fungi, 2) correct moisture and relative
humidity, 3) optimum temperature, and 4) sufficient time in storage.

Koehler (1938) reported Aspergillus grew at a minimum of 14.3%
moisture, Penicillium at 15.6 to 20.8 and Fusarium moniliforme at 18.4
to 21.2%. Trenk and Hartman (1968) concluded that rewet corn at mois-
tures below 17.5% and temperatures below 13 C was not susceptible to
aflatoxin fonnation by A, flaygs. From 19 to 28% moisture, toxin
content increased linearly from 50 to 2,000 PPB.

Diener and Davis (1967) reported the maximum temperature for

aflatoxin production was 41.5 C, the limiting relative humidity was

85% and minimum temperature was 13 C. Milner and Geddes (1946) con—
cluded the relative humidity rather than actual moisture determine
susceptibility to molding. The critical moisture values for differ—
ent seed species are those in equilibrium with a relative humidity
of about 75%.

Lutey and Christensen (1963) found that storage of barley at
15% moisture and 30 C for 16 weeks resulted in total loss of viability
of all field fungi and little invasion of Aspergillus. Landecker and
Stotzky (1972) found that when they grew fungi in the presence of bac-
teria, the bacteria strongly inhibited colony spread and sporulation.
Moreover, colony morphology was changed presumably by the release of
volatile metabolites.

Using casein substrates, Lie and Marth (1968) found A, flgygs
was able to initiate growth in the pH range of 1.7 to 9.34, with the
best growth between 3.42 and 5.47 mold growth was always associated
with a pH change toward neutrality. Both aflatoxins B1 and G1 were
detected in all samples which supported mold growth, but highest con-

centrations were noted at the pH extremes.

Documented Cases of
Mytotoxin Poisoning

When feeds are subjected to fungal growth, the fungi produces
metabolites which may be beneficial, harmful or have no effect on the
animals which consume the moldy feed. Christensen and co-workers

(1973) added a toxin-producing strain of Aspergillus flavus to grain

 

which was heavily infested with natural fungi. A small amount of
toxin was found in one sample, but feeding this grain caused no in-
jury in rats, ducklings or broiler chicks. This work suggests that
the danger of toxicity from materials invaded by a mixture of fungi,
including one strain of A, flgygg capable of producing toxin, is not
great.

Whiteher (1971), Doupnik and co—workers (1971) and Seerley and
co-workers (1972) fed corn infected with Helminthosporium mgygj§_and
found a complete lack of toxicity in swine, chicks, mice and rabbits.
They did note a decreased digestibility and cautioned that this
blighted corn was more susceptible to invading fungi during storage
and care should be taken to avoid secondary contamination.

In feeding an extract of Aspergillus grygg§_to sheep, Niver,
Tucker and Mitchell (1971) found no effect on fiber digestibility or
N balance and defaunation of the rumen was not detected. Hintz and

co-workers (1967) fed 450 PPB of aflatoxin B to pigs from 3 to 8

1

months of age with no harmful effects. A11 gilts and one boar were

10

continued on feed for a reproductive study which resulted in all off-
springs appearing normal at 6 weeks of age.
Lynch (1972) listed the main storage fungi and toxins produced:

Aspergilli - Aflatoxins, ochratoxins, patulin and
sterigmatocystin

Fusaria — Zearalenone, acetamido lactone, tricothecenes

Penicillia - Rubratoxins, patulin, citrinnin and tremorgenic
factors

Other - Slaframine, sporidesmins, ergots

Mirocha et a1. (1968) reported that when moldy hay was included

 

in the ration of 150 dairy cattle the number of services per conception
jumped from 1.2 to 4.0. An estrogenic factor was isolated (F—2) at a
concentration of 14 PPM. When cultured, Aspergillus sp, and Penicil-
ljgm_§p, were found but Fusarium would have died off at moisture con-
tents that permit growth of Aspergillus and Penicillium. Burt and co-
workers (1964) reported that when hay which was not visually molded
but had total spore counts of 2,000,000/g was included in the ration
of dairy cows, a 6% decline in milk yield was observed. Large numbers
of Aspergillus and Penicillium were detected in this hay.

Lynch et a1. (1969) reported a field case in which ensiled,

 

moldy, shelled corn produced tetany symptoms in milk cows. Of the
four cases which occurred, all responded to IV infusion of calcium

gluconate. Apparently this ration contained a normal level of minerals.

11

The most predominate fungi in the corn were yeasts, Mgggr§3Penicillia,
Monascus,and Byssochlamys.

Still gt_gl. (1971) isolated Aspergillus ochraceus from moldy
hay that was associated with abortions in dairy cattle. Ochratoxin
was isolated from a pure culture of the fungi and caused faetal death
in rats. Albright and co-workers (1964) diagnosed a gross hemorrhagic
syndrome in 20 to 29 heifers fed a ration containing toxin-producing

strains of Aspergillus flavus, Penicillium cyclopium and Penicillium

 

palitans. Smith and Lynch (1973) analyzed mold and unmoldy corn
silage which had been fed to a herd of cows in which several deaths
had occurred. They found A, fumigatus at dilutions of 5 X 106, but
analysis of feed for toxins by thin-layer chromatography was negative.
They concluded that the corn silage may or may not have been impli-
cated in the death of the cows.

Mohanty gngfl, (1969) molded alfalfa hay by sprinkling with
water. They hay quickly heated to 57-64 C. Molding lowered DM, ether
extract, and NFE, while increasing pH, ash and ammoniacal nitrogen.

In feeding trials, the molded hay reduced DM intake, BW gains, total
VFA, ruminal ammonia, and rumen protozoal counts; whereas, increases
in rumen pH, percent acetate and Diplodinium numbers were noted.
Steers fed the molded hay showed laxation of feces and developed
rough hair coats. Nineteen mold species were isolated with Sggpg:

lariopsis brevicalis predominant.

12

Mohanty gt_gl. (1968) in another report molded hay and extrac-
ted with various solvents. The chloroform extract gave the greatest
growth depression when fed to baby chicks. Aspergillus appeared to be
predominate fungal species in this study.

In ewes Cysewski and Pier (1968) produced abortions with IV
injections of spores from A, fumigatus. Chu and Chung (1971) reported

the L D in day-old chicks to be 166 and 216 pg for ochratoxin A and

50
C. No toxic effect was demonstrated when chicks were fed with up to
500 ug/chick of dihydroisocoumarin, the hydrolyzed product. Christen-
sen and co-workers (1965) found that 12 of 85 isolates of Fusarium
caused increases of 5-8 times in weight of the uterus of rats. These
isolates originated from feed collected on farms.

Kurtz gt_gl. (1969) reported gilts given estradiol (F-2)
mycotoxin and corn which had been inoculated with Fusarium graminearium
exhibited the same histological changes in the genital tract. These

were characterized by squamous cell metaplasia and loss of normal mu-

cosal epithelium off the vagina and cervix. Sharda et a1. (1971) fed

 

corn which had been inoculated with Fusarium rosium Ohio isolate C and

 

showed it Was highly toxic to rats, mice, hamsters, rabbits, and pigs.
Post-mortem examination revealed jaundice, histological changes in
liver and myocardial granulomas. Molds depressed feed consumption of
all animals but pigs. Permanent damage did not appear in rats and

replacement of moldy corn with good corn reversed symptoms.

13

Muller ejAEl. (1970) reported sensitivities of several animals
to aflatoxin. These animals in order of decreasing sensitivity were
ducklings, turkey poults, goslings, young pheasants and chicks. Sinn-
huber EILEl, (1969) has reported cancer formation in trout due to

aflatoxin consumption. Krogh et a1. (1970) isolated two nephrotic

 

compounds, citrinin and oxalic acid from Penicillium viridicatum.

In addition to these toxins, Crump et a1. (1963) reported iso-

 

lation of Rhizotonia leguminicola from hay known to cause slobbering
in cows. The isolate was grown in pure culture and fed to rats and

guinea pigs and produced similar symptoms. Rainey et a1. (1965) iso—

 

lated a salivary factor from a pure culture of A, leguminicola which

possessed parasympathomimetic action.

Toxicity and Secretion in Animals
Given a Known Amount of Mycotoxin

 

 

Lynch gt_gl. (1970) fed aflatoxin to six pairs of calves for
6 weeks. The calves exhibited a strong objection to feed containing
more than .020 mg aflatoxin B1. Weight changes, albumin/globulin
ratios and total serum protein content of calves were not affected,
but a significant increase in serum alkaline phosphatase occurred at
.020 mg BI/kg BW. At post mortem, livers showed a loss of color and

adrenal hyperplasia was noted. Histological studies of the liver

14

indicated bile duct and central vein proliferation, accumulation of
fat, and loss of glycogen. To avoid the feed refusal Lynch §t_gl.
(1971) dosed seven pairs of young dairy calves with 0 to .10 mg/kg BW
of GT aflatoxin for 6 weeks. Most of the aflatoxin-induced changes
occurred at the .08 and .10 mg doses by the second week of treatment.
These were decreases in feed intake, gains, serum carotene, inorganic
phosphorus, serum vit A, and increase serum alkaline phosphatase and
total bilirubin. At post—mortem, livers were again light tan with
bile duct proliferation at the .04 mg level. Gall bladders were 10
times their normal size.

Marth (1967) found rats excreted some aflatoxin as C02, some
in urine and feces, but 6-9% of ingested toxin remained in the liver.
In addition rats and dairy cattle can modify and excrete some toxin
in the casein fraction of their milk.

Masri et a1. (1967) fed a cow and a ewe aflatoxin B1 in their

 

diet. About 0.3% of the ingested aflatoxin appeared in the milk.

From 2 to 3 percent of the original aflatoxin dose appeared as M1 in
milk, and excretion was completed a few hours after ingestion. All-
croft and co-workers (1968) gave a lactating cow an oral dose of 300
mg mixed aflatoxins (B1, 44%; G], 44%; B2, 2%). Urine, milk and feces
was collected for 9 days. About 85% of the toxin secreted was de-
tected in 48 hours; however, only 4.5% of the original dose was re-

the urine 1.55% as M and 2.79%

covered. The milk had .18% as M], 1

15

was found in feces as B]. Some G1 was in both urine and feces.
Apparently the cow must metabolize the toxins to M1 before secretion.
The ewe reacts similarly to the cow in excretion of afla-
toxins. Nabney SELEJ: (1967) dosed a ewe with mixed aflatoxins (B1,
36%; G], 52%; B2, 3%; Ge, 2%). Ninety percent of that excreted
occurred during the first 48 hours. Only 8.1% of the dose was re-

covered. Milk had 0.1% as M1, urine 6.4% as M1 and G], and feces

1.6% as G1 and B].

Contamination of Human Food Supply Via Fungal
Toxin Consumption by Animals

In a survey of commercially available milk in the United

States no aflatoxin M], was detected (Brewington et a1., 1970). In

 

contrast Purchase and Vooster (1968) showed that 2 of 21 commercially
available milk samples in South Africa contained aflatoxin M1 in

easily detectable amounts, while 3 others contained traces.

16

Methods of Controlling Mycotoxins in Feeds

Methods of preventing mycotoxin contamination of the human
food chain are to destroy the toxins by chemical or physical means

or prevent the toxin—producing fungi from developing.

Destruction of Mytoxins by Treatment

Dollear gt_gl. (1968) heated peanut meal containing aflatoxins
in the presence of moisture and chemicals. They showed that ammonia,
methylamine, sodium hydroxide and ozone were effective in either
destroying or reducing aflatoxin levels. Burnside gngl, (1957)
found the toxicity of mycotoxins unchanged after heating to 60 to 70 C
for 26 hours. From these limited data, it appears that the most econ-
omical way to solve the mycotoxin problem is the prevention of fungal

growth by chemical or physical treatment.

Prevention of Fungal Growth with a Fungicide

As early as 1945 Snow and Watts (1945) tested 50 isolates of
molds for their reaction to sulphonilamide, sulphonamide E.O.S., sul—

phapyridine, sulphamezathine, sulphaquanidine, propamidine, and

17

phenamidine. They found sulphonamide doubled the storage life of

ground linseed cake. Milner et a1. (1947) tested the fungistatic

 

ability of 100 compounds on wheat stored at 16 to 25% moisture.

They found eight compounds which had satisfactory fungistatic prop-
erties. These compounds listed in order of their effectiveness are
8-hydroxyquinoline sulfate, thiourea, P-aminobenzoic acid, sulfonil-
amide, benezene sulfonamide, 2 aminothiozole chloramine B and calcium
propionate. They found a variation in effectiveness for different
fungi and suggested that a given compound should be tested on the
flora of the product which is to be treated.

Christensen and Gordon (1948) found chloramine B, spergon
dust, P—toluensulfonilamide or thiourea had very little fungicidal
activity when applied to wheat inoculated with A, jjgygs, Knodt gt
31;_(1952) reported sulfur dioxide treatment of wet forage produced
a very palatable grass silage. The silage had decreased surface
losses and spoilage rate during summer feeding.

Wittwer et a1. (1955) treated red clover and grass forage with

 

molasses, brewers dried grains and sodium bisulfite. Molasses-treated
silage was most palatable and sodium bisulfite the least. They con-
cluded that the small savings in nutrients could not justify the cost
of preservatives. Srinivasan and Majunder (1965) reported that fumi-
gation of kafir corn with methyl bromide and ethylene dibromide

destroyed mold and bacteria.

18

Wilcox (1972) published a list of common feed preservatives

and their characteristics:

Propionic acid

Acetic acid

Formic acid

Lactic acid

Sorbic acid

Insobutyric
acid

very effective and widely tested preservative
pungent odor
calcium and sodium salts appear less effective

about 50% as effective as propionic at 16 to
20% moisture levels and less effective over 25%

more effective as a preservative when combined
with propionic acid

much used in foods for pickling
pungent odor

salts of acetic acid have almost no preserving
effect

preserving effect somewhat less than acetic
acid

dangerously caustic to skin

very pungent odor

calcium formate used in silages

some preserving effect in high moisture feeds
needs further testing as a grain preservative

along with potassium sorbate is used in bakery
doughs to inhibit molds and yeast

effective grain preservative when dissolved in
alcohol and thoroughly distributed in the grain

reported to have preserving effect on grain

quite pungent odor

19

Benzoic acid - preservative for foods and fats
and
Sodium Benzoate - limited by law to less than .1% in foods and
feeds

needs further testing for grain preservation

Propyl-p-hydroxy - used in foods--1imited to not over .05%
benzoate

needs further testing for grain preservative
effects

It appears that several compounds could be used as fungicides
in feeds; but because of cost and legal restrictions the volatile
fatty acids and their salts offer greatest promise. If their use is
to become widespread they must not decrease the animal efficiency, but
they must preserve the feed. Literature concerning the effect of
these compounds on the animal and preservation of feeds will be re-

viewed.

Acid Treatment of Feeds and Grains
Acid Effect on Animal and
Feed Quality

Simkins, Suttic and Baumgardt (1965) infused VFA mixtures (60%
acetate, 20% propionate, 20% butyrate) into cows on pelleted alfalfa
hay rations to meet 15% of the estimated digestible energy requirement
and showed that infusion of propionic and butyric acids depressed feed

intakes. They concluded that VFA's can act as satiety signal compounds.

20

Also, Montgomery et a1. (1963) reported a significant decrease in hay

 

consumption after intraruminal acetic acid infusion, while the infu-
sion of propionic, butyric and lactic acids caused a moderate decrease
in voluntary intakes.

Ulyatt (1965) reported decreased feed intakes in sheep intra-
ruminally infused with 200 cal of acetic acid on low and high planes
of nutrition, but the decrease was more pronounced on the low plane.
Increased intakes resulted from infusing 200 cal. of propionic acid
on both planes of nutrition; but at 300 cal. propionic depressed con-
sumption at the low level of nutrition.

Bentley §t_gl. (1956) reported the addition of sodium salts of
acetic, propionic, and lactic acids to corn-cerelose-urea—hay or corn-
hay rations produced significant increases in gains of lambs. The
apparent feed replacement values were calculated at 1 kg of the acid
salt for 3-10 kg of feed. Armstrong and Blaxter (1957) reported that
the administration of 400 cal of acetic acid, propionic or n-butyric
or 800 cal as n-buryric to sheep in positive energy balance did not
interfere with the normal process of rumen fermentation or impose non-
physiological conditions upon the animals.

Balch and Rowland (1959) administered .5 to 1.5 kg sodium ace-
tate to cows on a fat-depressing ration and increased milk fat percen-
tages. Administration of 414 g sodium propionate did not restore the

fat percent and sodium acetate addition, to a normal diet,had no

21

affect on milk fat. Rook and Balch (1961) infused acetic, propionic
and butyric acids intraruminally and reported acetic acid caused an
increase in milk production and in yields of fat, lactose and protein,
as well as an increase in fat percentage. Butyric or propionic acids
had no effect on milk yield, but propionic decreased fat yields and
percent while increasing percent protein and solid non-fat; whereas,
butyric acid specifically increased the yield and percentage of fat.
Vercoe and Blaxter (1965) reported that the infusion of sheep
with fonnic acid ondried grass diets at a constant rate for 17 days
increased methane and CO2 production but there was no significant

change in 0 consumption.

2

Birdson (1972) reported 6% faster gains in steers fed HMC
treated with 1.5% acetic and propionic acids (60:40 ratio) compared
to dry corn. Feed efficiencies favored the acid-treated corn. Jones
fflijil. (1970) treated HMC with 1.5% propionic acid and ensiled other
HMC. When the treated corn was fed to dairy cows and heifers FCM,
persistency of milk production, milk fat and protein percent and rate
of gain were not significantly different for animals fed untreated
HMC.

Clark £3411. (1973) fed 50 cows dry ensiled or propionic—
treated corn with either hay or haylage. Cows fed ensiled and
propionic-treated corn produced more 4% FCM than cows fed dry corn.

In contrast to this Johnson and Otterby (1973) reported that

22

unadjusted milk production was higher on dry than high moisture or
acid-preserved corn.

McCaffree and Merrill (1968) reported two trials with dairy
cows in early lactation which were fed HMC. Feeding HMC resulted in
lower forage and total DM intakes, milk fat percent, but higher
actual milk production compared to feeding dry corn. Solids corrected
milk was not affected. Barrington and Jorgensen (1971) found milk
production and feed efficiencies were similar for HMC and dry corn,
but milk fat tests were lower for HMC. In contrast to the drop in
fat percentage usually seen with HMC feeding Johnson and Otterby
(1971) reported that milk fat depression was not a problem in rations
containing 33% HMC and corn silage.

Beeson and Perry (1958) found fattening beef cattle utilized
high moisture ground ear corn (32% moisture) from 10-15% more effi-
ciently than regular ground ear corn when grains were adjusted to
the same moisture. In agreement with this Burroughs (1971) reported
corn harvested and stored at 24-30% moisture had a feeding value on
a DM basis 4—9% higher than artificially dried corn. Wilson and Long
(1972) treated HMC with 1.6% acetic acid and propionic acids (60:40)
and observed a 5% increase in feed efficiency of steers compared to
steers fed dry—untreated corn. No significant differences in digesti-

bility were seen.

23

Bayley §t_gl. (1972) reported greater nitrogen and energy re-
tention for high moisture corn preserved by addition of organic acids
or by ensiling than dry corn in trials with pigs.

Bade 23:21: (1973) reconstituted dry sorghum to 70% DM with
either water or 2% acid. Coefficients of digestibility for all com-
ponents were highest for the water-reconstituted grain ration and
lowest for the dry grain ration. Fat corrected milk was not affected
by treatment but actual milk was highest on the reconstituted grain.

Bolsen et a1. (1973) tested ammonium isobutyrate, aureomycin, sodium

 

hydroxide and a mixture of acetic and propionic acids as forage sor—
ghum additives. The concluded that feeding values of forage sorghum
silage was not significantly improved by any of the four additives.
Marion §t_gl. (1972) fed steers HM sorghum grain treated with
O, 4, and 6% acetic or propionic acids. Steers gained well at the
lower levels of acid and gains were higher than controls at 4%, and
lower at 6% added acid. In another trial propionic acid was added
at 2% to dry or reconstituted grain (30% moisture) and stored for 14
days in open barrels. When compared to the untreated grain no sig—
nificant differences in daily gain, feed intakes, or efficiency were

observed.

24

Factors Affecting Silage Quality

Barnett and Duncan (1954) and Langston et a1. (1958) charac—

 

terized poor quality silage by pH values of 4.8 or above, low amounts
of lactic acid, high levels of butyric acid, and high ammonical nitro-
gen. Poor chemical quality and a lower rate of nutrient preservation
were associated with poor quality silage. They recommended compress-
ing the mass to make it more air tight.

Langston gjggl, (1962) observed aerated silages had high temp-
erature and pH values with increased butyric acid and depressed lactic
acid. They noted high total acids in sealed containers and concluded
that high levels of sugar did not insure silage of superior quality
unless the forage was packed properly to exclude air. Zimmer and
Gordon (1964) using laboratory silos sealed for 38 days or sealed
with the exception of aeration on days 1, 2, 3, and 6, reported

higher 0 consumption of unwilted, chopped silage during day l and 2

2
of aeration. Grinding the material improved total preservation and
reduced C02 and DM losses. A correlation coefficient of +.71 was ob-
served between CO2 production and DM losses, so they concluded aera-

tion resulted in poor preservation of the silage.

Wierginga et a1. (1961) concluded that the presence of oxygen

 

resulted in a faster loss of soluble sugars because respiration con-
tinues for a longer time. Above 40 C, oxygen was responsible for the

fixation of protein into indigestible compounds. This temperature

25

was also associated with the highest percent of butyric acid. They
recommended that silages be kept below 30 C to prevent putrefaction
and fixation of protein which makes it indigestible.

Lopez gt_gl, (1970) observed greater pH values for corn
silages at low (25%) and high (52%) than at medium DM (30%). A sig-
nificant decline in lactic acid concentrations was observed with ad-
vancing maturity. Total organic acids declined from 11.94% of DM in
25% DM to 3.14% of DM at 52%.

Ohyam §£;§lx (1973) investigated the effect of glucose and
temperature in grass silage preserved in laboratory jars. With no
additive silage at 30 C was of very poor quality while at 15 C qual-
ity was improved. Glucose addition resulted in excellent quality
silage irrespective of the temperature. Extent of protein breakdown
was affected by temperature at the early stages and by pH at the
later stages of ensiling.

Huber gngl, (1972) treated corn silage with formic, acetic,
propionic, and lactic acids at levels from .17% to .85%. Lactic acid
production was depressed by formic acid (from 10% of DM in control to
.9% on .85% formic), unchanged by acetic and increased at higher levels
of propionic and lactic acid. Acetic acid levels were higher than
control at .17, .34, and .57% formic, but 50% of the control at .85%.
Acetic acid treatment increased silage acetate when .68 and .85% was

added; whereas propionic and lactic acid additions decreased acetate

26

to about 50% of the controls. Huber (1970) also showed a greater de-
pression in lactic acid content resulted from formic acid treatment
of corn silage than has usually been reported in other crops (Castle
and Watson, 1970).

Carpintero gt_gl. (1969) treated lucerne with .85% formic
acid and found this level was sufficient to achieve an immediate pH
fall to 4.2. Formic acid inhibited both lactic acid and clostridial
activities. They also observed preservation of water soluble carbo-
hydrates (WSC) and suggested a beneficial effect on ruminants.
Wilkins and Wilson (1969) added formic acid at the rate of one half
gallon per ton to grass silage and detected an immediate drop in pH
to 4.4 with little change during the first 12 days. Lactic acid in
the treated silage was low.

Henderson and McDonald (1971) treated grass of low DM (11.8-
17.3%) with formic acid and found the acid prevented oxidation of WSC.
Treatment at .34% or higher also decreased proleotysis, lactic acid
production and volatile nitrogen.

Castle and Watson (1970) treated timothy and perennial rye
grasses (l7-20% DM) with 0.2% formic acid. Formic acid treatments
had about 7-15 C lower temperatures. Lactic acid was higher and
butyric lower in treated silages indicating that formic acid improved

silage fermentation. Waldo et a1. (1971) reported higher energy

27

recoveries from unwilted silage treated with formic acid compared to
no treatment.

Waldo gt_§l. (1973) found that dairy heifers grew better on
well made wilted silages than on direct cut silages treated with
formic acid. In contrast, Castle and Watson (1970) found wilted
silage was inferior to formic-treated silage for maintaining milk

production.

Effectiveness of Organic Acids in
Preventing Fungal Growth

Studies with Grains

Porter (1946) found organic acids had a greater inhibitory or
germicidal effect than mineral acids at the same pH which was attrib-
utable to the whole (undissociated) molecule rather than solely to
the hydrogen ion concentration. Weise (1971) tested the effect of
adding 0.1, 0.3, 0.5, 1.0, 3.0, and 5% propionic acid to agar media

on the growth of Candida, Pichia, Hansenula, Torulopsis, and mold.

 

All but Torulopsis (which required .5%) were completely inhibited by
.3% propionate. It was also noted that molding of cultures could be
prevented by adding .25% Na propionate, but the inhibition of molds
was greater than in yeasts.

Richardson and Halick (1957) showed that propionic acid or

propionic anhydride inhibited the growth of molds and heating in

28

corn meal at 0.1% addition. Calcium propionate was effective at 0.3%,
but no inhibition was shown for sodium propionate at .6%, or for pro-
pionamide or propionanalide at 0.3%. An acid environment was required
for maximum effectiveness of propionate. Butyric, valeric, and caproic
acids delayed heating when added at .1 to .2 percent and heat inhibit—
ing activity decreased as chain length increased. However, prevention
of mold appeared to increase with chain length. Sorbic acid also
appeared very effective in depressing heat production.

Despite the decreases in spoilage due to acid addition, the
authors (Richardson and Halick, 1957) concluded that drying was the
only practical way to properly preserve feeds.

Sprague and Breniman (1969) reconstituted dry cracked corn to
20, 25, and 30% moisture and ensiled in quart jars. They concluded
that the minimum moisture to provent mold in HMC was 30-33%. Drysdale
(1970) estimated losses due to production of C02 during fermentation
in sealed storage were as high as 5%. He suggested that the value of
feed lost was almost equal to the cost of adding propionic acid which
prevented C02 losses. The acid-preserved grain was found to keep for
a year or more under open storage conditions.

Miller (1971) reported the amount of propionic acid needed for
preservation was directly proportional to the moisture content of the
grain. For HMC at 25% moisture, 1% propionic acid was sufficient

while at 30% moisture 1.25% was necessary.

29

Jones gt_gl. (1970) detected no heating or mold growth in HMC
corn treated with three different mixtures of acetic, propionic, and
butyric acid after 7 days of storage in sealed bags; however, in 7
days the controls molded (185,000-62,000 calories/g) had an off odor
and heated. A pH of 4.4, which was obtained with 1% acid addition
to 72% DM ear corn, was effective in inhibiting mold fonnation during
6 months of storage. However, Jones (1971) reported HM ear corn
molded and heated within several weeks after treatment with 1.2%
propionic acid.

Arends SELELL- (1972) treated 27% moisture corn with 1.5%
acetic and propionic acids (60:40). Control corn was dried to 12%
moisture. Mold spore counts of the untreated dried corn were 4.6 X
105/gm while the counts of the treated dried and treated HMC were
6.04 X 102 and 23, respectively. Christensen (1973) showed that
treatment of sorghum (l6-l7% moisture) with 0.2, 0.4, or 0.8% pro-
pionic acid kept it free of molds for 483 days when held at 12 C.
Sorghum at 19% moisture which was treated with .1 and .2% propionic
at 27 C became heavily molded in 16 days. Corn (19-20% moisture)
treated with .5% propionic and stored at 25 C was free of fungi
after 54 days while samples at 30% moisture treated with 1.0% pro—
pionic plus acetic acids (60:40) were free of molds after 140 days
when held at 20 C. Samples treated with enough acid to prevent

molding had zero germination. Alfalfa pellets (16% moisture)

30

treated with .5% and .8% propionic acid had no molds after 210 days
at 20 C. Other reports of prevention of molding in feeds stored for
animals are by Jones gtggl, (1970), Marion ££;213 (1972), and Young
gijgL. (1970).

Singh-Verma (1971) found propionic was more effective and
longer lasting if added after deterioration of grain had already
started because cells were growing in their log phase. Propionic
acid was highly biocidal to saprophytic cocci, coliform bacteria,
Aspergillus, Mgggg, Rhizopus, Alternaria, and a variety of yeast.
The growth of Bacillaceae, Actinomyces, Penicillium, Fusarium, and
Cladosporium was also strongly inhibited. In contrast, Sauer gtJiL.
(1972) found higher levels of propionic or acetic were required when
fungal invasion had already started. Fungi which grew in grain
treated with insufficient amounts were the same type as in untreated

grain.

Studies with Forages

Refermentation (spoilage upon removal from the silo) is often
a serious problem to cattle feeders. With a clover-grass mixture
Daniel fflijil. (1970) showed that propionic treatment reduced the fre-
quency and intensity of refermentation. Hillman and Thomas (1973)
found the addition of .4 to .6% propionic acid reduced molding of

haylage in small silos when ensiled at 40% moisture.

ll
1
’l‘

31

Sleiman et a1. (1972) treated whole chopped corn with various

 

mixtures of formic, acetic, and propionic acids. All acids reduced
temperature increases in silos. Days until molding were highest for
propionic and lowest for control forages. Dry matter discarded was
highest for acetic and lowest for acetic plus propionic treatments.
All forages were readily consumed by growing heifers. It was con-
cluded that the most effective retardation of spoilage was observed
from adding propionic at 1% or higher.

Asplund (1971) reported that a mold inhibitor added to hay
baled at 30% moisture prevented poor digestibility and acceptability
usually associated with mold growth. Sleiman (1972) concluded that
propionic acid could be used to prevent extreme losses of forages
stored under minimal protection.

Henderson et a1. (1972) found yeasts were more active in

 

formic acid treated herbages than in untreated material. Losses due
to fermentation plus oxidation in wilted silages were higher in the
acid-treated than untreated silage. Total microbial counts were lower
on formic acid treatment. Taylor and Phillips (1970) also found that
total aerobic counts were lower, while lactate fermenters and thermo-
philic counts were higher in formic acid treated than untreated

silages.

32

Non-Protein-Nitrogen Treatment of
Forages and Grains

Effects of Ngfl-Protein-Nitroggfl
on Fermentation and Animal

 

Schmutz £3411. (1962) ensiled HMC at 24, 32, and 45% moisture.
Lactobacilli and anaerobes were 109 per gm after 10 days for all
treatments and yeasts numbered 109 for the 24% and 108 for the 45%
corn at 60 days. Acetic acid was highest at 45% moisture. Lactic
acid concentrations of the HMC were directly related to moisture con-
tent as were weight gains in growing heifers. When urea was added to
the HMC 50% was degraded to ammonia by 20 days. Schmutz §t_gl. (1964)
noted that high moisture ear corn with added urea was initially lower
in pH than untreated corn but this reversed during fermentation.
Lactic acid was increased and acetic decreased with the addition of
1% monobasic calcium phosphate to HMC (Schmutz, £3411. 1964).

Dutton and Otterby (1971) found that HMC treated with diam-

monium phosphate had higher pH values, NH levels and greater nitrogen

3
losses than controls or that treated with soybean meal; whereas the
urea treatment was highest in acetic and lowest in propionic acid.
Huber and Santana (1972) found that dairy heifers ate more
ammoniated than control silage when these were the only feeds offered.

Also, lactating cows produced more milk on ammonia or untreated silages

than on a negative control ration but no differences between the

33

non-protein-nitrogen treatments were noted. Ammoniated silage had
higher lactic acid and water insoluble nitrogen than urea or control
silages.

Webb gfl;i{1. (1972) reported intraruminally-administered ammon—
ium acetate gave greater rumen ammonia concentrations than did isoni-
trogenous quantities of urea. Urea elevated rumen pH but ammonium
acetate did not. Both NPN supplements increased concentrations of
ammonia and urea in peripheral blood. To produce toxicity it took
less nitrogen from urea than ammonium acetate.

Allen et a1. (1972) found no difference in gains of fattening

 

steers supplemented with soybean meal, liquid urea, 1/2 liquid urea
and 1/2 corn steep water, ammonium (NH4) formate, NH4 acetate, NH4
propionate, NH4 lactate, or NH4 butyrate. Dutrow and Huber (1973)
used urea, ammonium propionate, ammonium lactate or soybean meal to
supply 80% or more of the supplemental nitrogen in high corn-corn
silage rations for dairy cows and showed that fat corrected milk was

significantly higher for the NPN than soybean meal diets. Dry matter

intakes were not different between treatments.

Effect of Non-Protein-Nitrogen
on Fungal Growth

Tomkins and Trout (1931) found green rot of citrus due to

Penicillium digitatum was greatly reduced by storage in an atmosphere

34

of 500 to 1000 ppm ammonia. This concentration was produced by damp
crystals of ammonium bicarbonate while the release of ammonia from
ammonium acetate was not sufficient to prevent the rot. Tomkins
(1932) reported that ammonia increased the latent period of fungal
spore germination and that the same concentrations were needed to
inhibit germination as growth.

Altschul gt_gl. (1946) treated cottonseed with ammonia to in-
hibit plant respiration of mature seeds. They concluded that most of
the deterioration which occurs in stored cottonseed is due to the
action of enzymes in the seeds rather than microbial activity.

Bothast gt_gl. (1973) treated 26 or 12% moisture corn with
ammonia at 2 or 0.5% of the dry weight. Both concentrations of
ammonia eliminated external and infecting molds and yeasts, and
tended to reduce bacterial counts. Molds killed were species of
Aspergillus, Penicillium, Fusarium, Trichoderma, and Rhizopus.
Fusarium comprised 75% of external mold population while the rest

were Aspergillus and Penicillium. Original corn had 7.9 X 104 molds

 

 

and yeasts per gm, but after 24 hr. of tempering these increased to
5.1 X 105 per gm. Mice showed no preference for the tempered over

the 2% ammoniated corn.

35

Conclusion

 

After reviewing the literature it becomes apparent that the
growth of fungi in feeds during storage is a dangerous and expensive
problem. Addition of propionic acid has been shown effective in re-
tarding spoilage of high moisture grain. The effect of acid on
fungal growth of forages has not been studied.“ Ammonia treatment
of grains might also offer a less expensive preservative than organic
acids, but further research on levels, animal acceptance, and methods
of application is needed.

Little work has been done on patterns of fungal growth during
referementation and the effectiveness of organic acids or ammonia in
preventing losses during this period. Therefore, the objectives of

this thesis are:

a) to evaluate the types of fungi during storage and refermen-

tation of corn silage and HMC;

b) to determine the effects of short chain organic acid and
ammonia additions to corn and corn silage on fungal de-

velopment during storage and refermentation;

c) to ascertain the response of lactating cows to ammoniated

corn grain.

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Young, L. G., R. G. Brown, and 8. A. Sharp. 1970. Propionic acid
preservation of corn for pigs. Can. J. Animal Sci. 50:711.

Zimmer, E., and C. H. Gordon. 1964. Effects of wilting, grinding,
and aerating on losses and quality in alfalfa silage. J.
Dairy Sci. 47:652.

III.

PART A: FUNGAL GROWTH DURING FERMENTATION

AND

REFERMENTATION OF ORGANIC ACID TREATED

CORN SILAGE

JI__

Abstract

Chopped corn (35% DM) in lots of 56 kg was treated with either
propionic (P), formic (F), propionic + formic (PF), or propionic +
acetic (PA) acids at either 0, 0.5, 1, or 2%. Each treatment and
level was duplicated. Treated material was placed in polyethylene
bags inside 200 t drums and evacuated. During fermentation forages
were sampled and temperatures measured at days 0, 3, 5, 15, 20, and
40. On day 40 when fermentation was complete, 12 kg portions of the
silages were placed in Open containers at 25 C. Samples were taken
temperatures measured, and silages were aerated at days 0, 2, 14, 22,
29, and 36 to determine the effects of organic acids on refermentation
of corn silage. Samples were analyzed for VFA, lactic acid, pH, and
number and type of fungi.

Acid treatment at the 2% level reduced the average tempera-
ture when compared to controls (19.8 vs 24.4; P < .01) and .5 and 2%
acid reduced (P < .01) the maximum temperature compared to controls
during refermentation (33.6, 29.6, and 37.5, respectively). Propionic
acid was more effective than other acids at .5 or 1% treatment. Lac-
tic acid production was significantly depressed by 2% acid. All
treatments containing propionic acid required more days (P < .01)

48

49

before visual molding (7.0, 13.4, 20.0, and 20.5 for F, PF, PA, and P,
respectively). Days until complete spoilage was increased by acid
treatment. All acids significantly decreased fungal colonies within

2 days after addition. During refermentation all treatments at the 1%
level or lower exhibited a rapid increase in number of colonies; how-
ever, P showed a slower increase. The proportion of yeast was greatest
at initiation of fermentation and decreased at day 40. During refer-

mentation yeast started growth again. Geotrichum increased at day 40

 

of fermentation but was constant at other times. Aspergillus was sig-

 

nificantly higher at day 40 of fermentation and 36 of refermentation.

No significant amounts of Penicillium were detected at any date.

Introduction

 

Because of recent advances in synthesis of organic acids they
have become economically available for use in preserving forages by
preventing oxidation losses and fungal growth. Castle and Watson
(1970) treated timothy and perennial rye grasses with .2% formic acid
which had about a 7-15 C lower temperature increase than the un-
treated. Weise (1971) found that .3% propionic acid added to agar
media retarded the growth of yeats and molds. Richardson and Halick

(1957) inhibited the growth of molds and heating in corn meal with

 

50

propionic acid. Yeasts were reported higher in formic acid treated
herbages (Henderson gt_gl. 1972). Daniel £3431, (1970) reported a re-
duction in frequency and intensity of after fermentation when a clover-
grass mixture was treated with propionic acid. Sleiman gt_gl. (1972)
reported propionic acid at 1% was effective in retardation of spoilage

in chopped corn. Sleiman (1972) also concluded that propionic acid

 

could be used to prevent extreme losses of forages stored under mini-
mal protection.

Fungi in forages have also been reported to cause tetany in
milk cows (Lynch gtggl, 1969). Still gt_§l, (1971) isolated Aspergil-

lus ochraceus from molding hay that caused abortions in dairy cattle.

 

Albright gt___a_1_. (1964) diagnosed a gross hemorrhagic syndrome in 20 of
29 heifers fed a ration containing toxin-producing fungi and Mohanty
SflLél: (1969) fed molded alfalfa hay to ruminant animals and found re-
duced DM intake, body weight gains, total VFA, ruminal ammonia, and
total rumen protozoa.

The object of this experiment was to determine fungal growth
and types of fungi during fermentation and refermentation in chopped
corn forage after treating with different levels and combinations of
organic acids. Changes in acid production, temperature, pH and dry

matter in silages were also ascertained.

51

Materials and Methods

 

Whole corn plants (35% dry matter) were field chopped and
brought to a storage area where 56 kg portions were treated with
propionic acid (P), formic acid (F), acetic plus propionic acid (PA),
and formic plus pr0pionic acid (PF) acids at 0, 0.5%, 1% or 2% of the
wet weight.a The PA acids mixture contained 80% propionic and 20%

acetic acid while the PF treatment was 60% propionic and 40% formic

 

acid. For treatment, the correct amount of chopped corn was weighed
on a polyethylene sheet, and the acid was sprinkled on the surface
while mixing with shovels. The entire mass was then rotated several
times inside the polyethylene sheet to insure complete mixing.
Treated material was then sampled and transferred to two
200 2 metal drums, lined with polyethylene bags (5 mils thickness).
The bags were then evacuated using a vacuum cleaner, sealed, and
stored (drums with bags inside) in an enclosed barn. Additional
samples were collected on days 1, 5, 10, 15, and 40 by opening the
bags, quickly removing .5 kg portions, re-evacuating and sealing.
Samples were immediately frozen and stored at -20 C. While the bags
were open temperatures were measured with mercury thermometers in-

serted into the center of the silage mass.

 

aAcids supplied by Celanese Chemical Co., Corpus Christi, TX.

52

At day 40 of fermentation the barrels were reopened and 12 kg
portions were placed in open polyethylene containers and stored at
25 C. The remaining ensilage was weighed and discarded. Exposed
silages were aerated by transferring from one container to another on
days 1, 2, 3, 5, 7, 10, 14, 22, and 36 and sampled on days 2, 14, 22,
29, and 36. Aeration was done to maximize the re-fermentation rates
of silages.

After thawing at room temperatures, forages were analyzed for
DH, pH, lactic acid, and number and type of fungi. Dry matter (in
duplicate) was determined by placing approximately 50 g wet forage in
a forced-air oven at 90-100 C for 24 hours. Silages were prepared for
pH, lactic, VFA, and fungal analyses by homogenizing 40 g in a Sorvall
Omni-mixer.a The homogenizer cup was immersed in ice. Approximately
50 ml of the homogenized material was divided into two aliquots,
one for pr determination and the other placed in a container for
plating to estimate fungal populations. Extracts from the remaining
material were strained through two layers of cheesecloth and deprotein-
ized with 50% sulfosalicylic acid (1 part SSA to 10 parts extract).
The deproteinized extract was then centrifuged at 15,000 rpm for 10
minutes and the supernatant was stored in a freezer until analyzed

for lactic acid and VFA.

 

aIvan Sorvall Inc., Newton, Conn.

bE. H. Sargent and Co., Chicago, 111.

“I: Tr mu «Ala-.w
\
. I

53

Colorimetric procedures of Barker and Summerson (1941) were
used to determine lactic acid and VFAs were analyzed by injecting 3 pt
of the deproteinized sample into a Hewlett Packard F & M gas chromato-
grapha using a glass column packed with chromasorb 101 (80/100 mesh).b
The injection-port temperature was set at 340 C, the column at 285 C,
and the flame detector at 320 C. Nitrogen was used as the carrier gas 1 3
and flow rate was 30-40 mls per minute which created a retention time

-1

of approximately 7 minutes per sample. Sample VFA concentrations

 

were calculated by comparing peak heights with a standard solution
made with known weights of analytical grade acids in a stock solution
and diluted until concentrations comparable with samples were reached.
Fungal population was determined by transferring with a ster-
ile wide-mouth 10 ml pipette, 1 ml aliquots of the freshly homogenized
silage sample into a dilution bottle filled with 99mls of sterile,
distilled water. The sample was thoroughly mixed and serially di-
luted until the proper concentrations of fungal spores and mycelia
were reached (approximately 20-50 colonies per plate). Either l or
.1 ml of the diluted sample was then dispensed into sterile plastic
petri dishes. Enough potato dextrose agarc with 100 mg per liter of

novobiocin which had been melted and cooled to 45 C, was then added

 

aHewlett Packard F & M Scientific Co., Mocel 402.
bJohns-Manville, Celite Div., Denver, Colorado.

cBBL, Cockeysville, Maryland.

54

to cover the bottom of the petri dish and swirled to insure complete
mixing of the agar and silage homogenate. After cooling, the plates
were sealed and placed in the dark at 20 C for 7-10 days at which time
the plates were removed and colonies were counted using a colony

countera and identified according to genus.

Results and Discussion

 

 

Heating of treated and control silages was negligible during
fermentation and silage temperatures were usually within 1 to 2 de-
grees of ambient. One explanation for the lack of temperatures might
be the anaerobic storage conditions after evacuation. However, due to
the small mass of silage (56 kg) and the dramatic change in ambient
temperatures (20 C on day l to 7.6 C on day 20) small changes in
silage temperature might have been dissipated or overshadowed. Honig
(1969) found DM losses increased linearly with the amount of added air
and Federson (1971) reported oxidation was accompanied by large rises
in temperature, but temperature increases in silos without oxygen
present were negligible. Zimmer and Gordon (1964) found aeration in-

creased CO2 and DM losses. Complete anaerobiosis was apparently

 

aFisher Scientific Co., New York, N.Y.

55

achieved in this experiment as indicated by the negligible DM losses
which analyzed less than 3% on control silages.

Refermentation is very critical during feeding of a stored
feed such as haylage, HMC, or silage. In addition to the loss of
nutrients due to refermentation (aerobic fermentation on removal from
the silo) as discussed by Daniel g£_gl. (1970), toxic fungal growth
may also occur. Losses or damage of a feed from the time of exposure
to air until its consumption are not generally known but they are
largely dependent on environmental conditions. For these reasons the
influence of organic acids on refermentation was evaluated. Because
fungal growth is closely associated with heating of stored feeds
(Gilman and Barron, 1930; Milner and Geddes, 1946; Milner and Geddes,
1945; and Federson, 1971) measurements of both parameters were measured.

Aeration caused a marked increase in temperatures as shown
earlier by Federson (1971). Average temperatures (Table 1) during
refermentation were depressed at all levels of addition for all acids,

TABLE l.--Effect of Level of Organic Acid Addition on Temperature
Development in Corn Silage During Refermentation

 

 

 

LEVEL OF ACID TEMPERATURE MAXIMUM TEMPERATURE
% (AVERAGE e) (c)
0% 24.4A 37.5A
0.5% 20.8AB 33.60
1.0% 20.2AB 35.7AC
2.0% 19.8B 29.63
ABC

Means not sharing same superscript are different (P < .01).

 

56

but significantly so (P < .01) at 2.0%. Maximum temperatures during
refermentation were also less for all acid additions, but the decrease
was significant (P < .01) at the 0.5 and 2.0% levels. This agrees
with the work of Daniel g£_gl, (1970) who found propionic acid treat-
ment reduced the frequency and intensity of after-fermentation.
Richardson and Halick (1957) also found propionic acid inhibited heat-
ing in corn meal and Sleiman (1972) reported formic or acetic plus
propionic acids reduced temperatures of unprotected rye forage.

No significant differences were found between acid treatments
with respect to heating. However, all but one series of treatments
contained propionic acid and that series was treated with formic acid.
Castle and Watson (1970) found formic acid—treated silages had a 7-15 C
lower temperatures during fermentation than controls. It is difficult
to explain why the .5% level of acids significantly reduced maximum
temperatures and 1% did not.

Lactic acid concentrations (across all dates and treatment
levels) in silages (Table 2) were significantly decreased (P < .01)
by 2% addition of all acids, (2.54, 1.79, 1.56, and .65% of DM for
0, .5, l, and 2% acid additions, respectively). This indicates a
severe inhibition of microbial activity by acids during both fermen-
tation and refermentation. However, of more interest would be the
peak level of lactic acid which would be similar to silage which

would be fed from the silo. Lactic acid concentrations were

57

 

 

 

 

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58

apparently maximal after 15 days of fermentation which agrees with
Langston gt_gl, (1958).

Lactic acid on days 20 and 40 of fermentation for the control
silage was 5.8% of the dry matter (Table 2 and Figure 1). At .5% pro-
pionic or propionic plus acetic treatment, lactate was depressed to
4.0% or about 70% of the controls. Strong inhibition of lactic acid
production was observed on both .5% treatments containing formic acid.
This different affect of the acids when applied at the same rate may
be due to the relative strengths of the acids (Carpintero gt_gl.
1969). The pH (Table 3) reflect this difference with the .5% pro-
pionic and propionic plus acetic acids having pH's of 4.80 and 4.73
while the two formic acid treatments averaged 4.47. This finding is
in general agreement with Huber §t_gl, (1972) who observed marked de-
creases in lactic acid of normal corn silage treated with formic acid
at .3 or .6%, but smaller decreases with added acetic or propionic
acids.

All acid treatments at the 2% level completely inhibited
lactic acid production during fermentation due to the low pH's caused
by treatments (Table 2 and Figure 2). The pH's were 3.28 and 3.65
for the formic and formic plus acetic and 4.20 and 4.23 for the pro-
pionic and propionic plus acetic.

During refermentation lactic acid decreased markedly on the

control, .5% pr0pionic and .5% propionic plus acetic while a smaller

 

59

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decrease was observed on the formic treatments (Figure 1). After 30
days of refermentation a marked increase of lactic acid occurred on
the .5% pr0pionic, .5% formic, and .5% propionic plus formic. On the
2% formic treatments sharp increases in lactic acid were noted at the
beginning of refermentation (Figure 2). These increases preceded
those on the propionic treatments by about 3 weeks. The formic acid
was being removed from the treated material faster than the propionic

acid which permitted an earlier lactate production. This is substan-

 

tiated by the rapid increase in pH to 4.70 at 1% formic treatment on
day 2 of refermentation while the pH of the 2% propionic remained
at 4.28 (Table 3).

0n treatments showing low lactate levels during fermentation,
the pH and lactate began to increase considerably during refermenta-
tion. The water soluble carbohydrates were preserved by the acid
treatments, but once the pH increased, lactate-producing bacteria
began to proliferate. Apparently the lactate was metabolized during
later stages of refermentation (Table 2). A similar pattern was re-
ported by Sleiman (1972) who found that after complete spoilage lactic
acid was not found in control or acid-treated silages.

Acetic acid production follows the pattern of lactic acid.
Production at day 20 was decreased by .5% formic and formic plus pro-
pionic treatments and was inhibited by all treatments at 1% or greater

addition except the treatment which contained acetic acid (propionic +

 

63

acetic). Although fermentation was inhibited by 2% added acid as in-
dicated by lactic acid (Figure 2) the added acetic acid increased the
measured level comparable with control. During refermentation, acetic
acid increased most in silages which had not been fermented, and at
spoilage a general decline was noted. (Table 4)

Propionate (Table 5) and pH (Table 3) were not statistically
analyzed because the large acid additions exerted strong effects on
both parameters. The pH data (Table 3) substantiate those of Carpin-
tero g§_gl. (1969) who reported the strongly acidic nature of formic
acid which in this experiment, decreased pH to 3.28 at 2% addition
compared to 4.20 for 2% pr0pionic addition. Propionic acid production
(Table 5) was low on all treatments and increases on the propionate
treatments reflect that propionate added,which agrees with Sleiman
(1972).

Correlations between propionate, pH, temperature, and spoilage
were determined (Table 6). Average and maximum temperatures were posi-
tively correlated (P < .01) with pH. Levels of treatment and maximum
temperatures were negatively correlated (P < .01). Days until visual
fungi and days until spoilage were negatively correlated with pH (P <
.01). In addition a high positive correlation of .836 (P < .01) was
found between propionate and days until spoilage. Hence, the pH in-
creases during refermentation were accompanied by spoilage; but

whether this change in pH is the cause or result of fungal growth

TABLE 4.--Acetic Acid ContentA (% DM) of Organic Acid Treated Corn

64

Silage During Fermentation and Refermentation

 

 

 

 

FERMENTATION REFERMENTATION
O 3 5 15 20 40:0 2 14 22
CONTROL .39 .95 1.09 1.32 1.43 .84 .51 .45 .16
.5% P .35 .46 .42 .25 1.27 .89 .44 .07 .08
.5% F .37 .50 .20 .50 .49 .59 ..56 .20 .35
.5% P+F .35 .40 .22 .26 .54 .79 .80 .79 .02
.5% P+A .75 .82 .60 .88 1.36 1.97 .40 .81 .45
1.0% P .27 .48 .26 .26 .36 .68 .03 .76 .70
1.0% F .45 .65 .24 .26 .41 .57 .23 .97 .13
1.0% P+F .34 .50 .31 .38 .38 .48 .69 .42 .58
1.0% P+A 1.14 .21 .75 1.36 .52 .74 .85 .79 .83
2.0% P .27 .41 .41 .54 .48 .78 .61 .68 .06
2.0% F .48 .23 .65 .36 .69 .86 .72 .21 .17
2.0% P+F .44 .62 .25 1.33 .40 .67 .97 .62 .51
2.0% P+A .98 .23 1.08 1.47 1.19 1.89 .15 .50 .31

 

AEach treatment is the mean of two duplicates.

65

TABLE 5.--Propionic Acid Content (% DM) of Organic Acid Treated Corn
Silage During Fermentation and Refermentation

 

 

 

 

 

FERMENTATION REFERMENTATION
O 3 5 15 20 40:0 2 14 22
CONTROL .27 .25 .23 .12 .43 .27 .35 .36 .30
.5% P 1.18 1.59 1.20 1.27 1.27 .58 2.10 3.26 3.10
1% P 2.37 3.57 2.82 2.63 1.84 1.84 3.70 3.02 4.10
2% P 4.09 3.03 4.84 4.14 4.12 3.38 4.71 5.78 7.48
.5% F .40 .46 .36 .42 .29 .44 .36 .87 1.40
1.0% F .20 2.05 .09 .38 .12 .28 .24 .44 .44
2.0% F .09 .50 .25 .19 .38 .21 .21 .29 .23
.5% F+P .80 .99 .58 .68 .60 .59 1.14 1.23 1.51
1% F+P 1.46 1.68 1.50 1.51 1.23 .91 2.61 1.32 .86
2% F+P 5.89 4.59 3.44 3.46 2.92 2.72 5.22 4.19 5.25
5% A+P 1.76 1.25 .91 1.07 .98 1.10 1.70 1.24 1.70
1.0% A+P 3.19 2.54 2.14 2.10 1.63 1.64 3.10 2.63 3.03

2.0% A+P 3.58 4.08 3.61 4.09 3.48 3.53 3.79 3.98 3.61

 

 

66

TABLE 6.--Correlations of Several Variables During Fermentation and
Refermentation of Organic Acid Treated Corn Silage

 

 

VARIABLES r LEVEL OF SIGNIFICANCE
PH: Temperature .530 .01
PH: Fungi .701 .01
PH: Maximum Temperature .717 .01
PH: Days until Visual Fungi -.691 .01
PH: Days until Spoilage -.583 .01
Propionate: Days until Spoilage .836 .01
Level: Maximum Temperature -.613 .01

 

is not known. The high correlation between days until spoilage and
propionate confirms its action as an effective fungicide.

As the level of acid addition increased days until visual
fungal growth and days until complete spoilage also increased (P <
.01; Table 7). This agrees with Sleiman (1972) who found acid treat-
ment delayed spoilage of corn silage. Propionic acid was more effec-
tive than formic acid in retarding spoilage. Addition of formic to
propionic acid decreased the effectiveness of the propionic acid as
a fungicide as shown by 13.4 vs 20.5 days until visual fungi appeared,

but this was not observed with the propionic: acetic mixture. These

17,1.

 

67

TABLE 7.--Number of Days until Fungi Were Noted on Corn Silage,
Complete Spoilage and DM Loss During Refermentation as
Affected by Level and Type of Acid

 

 

 

LEVEL OF DAYS UNTIL VISUAL DAYS UNTIL
ACID FUNGAL GROWTH COMPLETE SPOILAGE
0% 5.0A 18.0A
.5% 7.5A 29.8B
1.0% 19.9B 34.3B
2.0% 28.5B 32.5B
ACID
TREATMENT
Propionic 20.5A 28.8
Formic 7.0B 26.3
Propionic + Formic 13.4C 29.8
Propionic + Acetic 20.0A 29.8
ABC

Means not sharing same superscript are different (P < .01).

data agree with Richardson and Halick (1957) who found propionic acid
a very effective fungicide.

Addition of 2% acid reduced (P < .01) DM losses during fermen-
tation compared to the three lower levels of acid addition. Losses
(as % 0f the original DM) averaged 1.5, 1.5, 1.3, and .5 kg for 0, .5,

l, and 2% acid addition, respectively. Apparently, this was due to the

68

nearly complete inhibition of fermentation by all acids added at 2%
0f the treated silage.

The literature revealed no reference to a method for quantifi-
cation of fungi in forages. A comparison of media with additives was
used for the identification and enumeration of fungi using corn
silage homogenized in distilled sterile water (Table 8). The media

TABLE 8.--Number of Fungal Colonies (per gm x 105) on Different Media,
with or without Novobiocin and/or Rose Bengal

 

 

 

 

MEDIA
PDA MALT CHRISTIANSEN'S CORN 'T
MALT MEDIUM MEAL
NO ADDITIVE 49 57 0 48 51
Rose BengalA 23 22 0 14 19
NovobiocinB 19 16 0 18 17
R°Se Bengal 16 17 0 20 18

Novobiocin

 

A33.3 mgs per liter of media.

B100 mgs per liter of media.

compared were: potato dextrose agar (PDA) (formulated to identify
fungi because of its low pH); malt agar; corn meal agar and Christen-
sen's medium (Christensen, 1946). All tests were conducted with the

following: 1) no additive, 2) rose bengal, 3) novobiocin, or 4)

 

69

rose bengal plus novobiocin. Rose bengal was used to retard the
growth of Mgggr_sp. but after determining insignificant amounts of
this contaminant present its use was discarded. Novobiocin was added
to retard bacterial growth. Christensen's medium appeared unsatis-

factory and slightly higher counts were obtained on the PDA than the

 

corn meal and malt agar.

Corn silage contains a wide variety of fungi during fermenta-

 

tion and refermentation. PDA is used to identify many types of fungi. 1 ‘
Because of this and slightly greater numbers of fungi isolated, PDA H
was chosen as the medium used in this study. Novobiocin was used be-
cause of its bactericidal properties. The apparent higher counts on
the medium which did not contain novobiocin was because bacterial
colonies were contaminating the fungal colonies and were being in-
cluded in the fungal counts (Table 8). No differences in types of
fungi isolated were seen between PDA, corn meal or malt extract. The
same type of colonies were also observed when inoculum was sprinkled
on solidified agar.

Total fungal colonies represents the approximate number of
fungi present in the samples. By the methods used it was not possible
to differentiate between colonies grown from mycelia or spores. It is
quite possible that all spores or mycelia did not grow; however, for

comparative purposes this procedure was satisfactory.

7O

Fungal colonies (Table 9) in green chopped corn were signifi-
cantly decreased (P < .05) within one hour after acid addition. The
fungal population of all silages decreased during fermentation, which
agrees with Federson (1971). Upon aeration (refermentation) fungal
colonies increased significantly (P < .01) and continued to increase
until day 36 of refermentation (Table 10 and Figure 3). Acid addition
across all dates and treatments significantly reduced the number of
fungal colonies obtained in plates (Table 11) which agrees with data
of Sleiman (1972).

For acid treatment of silage to be economical it must be
effective at an application rate of 1.0% or less. Fungal population
in material treated at 1% (Figure 3) reached a low level during fer-
mentation. With the exception of the propionic plus formic acid
treatment, all material treated with propionic acid or a mixture of
propionic had a marked lower rate of fungal growth during refermen-
tation than formic or control. Propionic alone had a slower rate of
fungal growth. On day 36 at the .5% treatment level (Table 9) pro-
pionic acid and propionic plus acetic appeared more effective than
the two treatments containing formic acid. Formic acid alone was
very ineffective as a fungicide even at the 2% level.

Propionic acid was present after 22 days of refermentation
(Table 5) so the increases in fungi were due to an adaptation to pro-

pionic acid and not its disappearance. The formic disappeared at a

 

71

 

 

 

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72

TABLE 10.--Number of Fungal Colonies Obtained from Organic Acid Treated
Corn Silage as Affected by Date

 

NUMBER OF COLONIES

 

DAY (103/gm)
FERMENTATION
1 234A
40 31A
REFERMENTATION
14 4,789B
36 21,387c

 

 

ABCMeans not sharing same superscript are different (P < -01)-

TABLE ll.--Number of Fungal Colonies as Affected by Level of Acid

 

 

Addition
NUMBER OF COLONIES
LEVEL OF ACID . (103/gm)
0% 22,486A
0.5% 3,286B
1.0% 384B
2.0% 284B

 

ABMeans not sharing same superscript are different (P < .01).

73

 

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74

faster rate than the propionic as indicated by the pH data (Table 3)
so the fungicidal effect of formic whether it be pH or the molecule
itself is lost at a faster rate than propionic acid.

The proportion of yeast (Table 12) was highest at initiation

of fermentation and decreased at day 40 while Geotrichium increased

 

during fermentation. During refermentation yeast growth was again

accelerated but depressed near the end of refermentation. Aspergillus

 

was highest on day 40 of fermentation and 36 of refermentation. No

significant amounts of Penicillium were detected at any date.

 

TABLE lZ.--Types of Platable Fungal Colonies at Day 1 and 40 During
Fermentation and Day 14 and 36 During Refermentation.

 

 

 

 

DAY YEAST GEOTRICHUM ASPERGILLUS sp. PENICILLUM sp.
1 FERMENTATION 71.7%C 17.3%A 0.1%A .04%

4o FERMENTATION 8.4%A 35.2%B 50.5%B .07%

14 REFERMENTATION 65.3%C 26.7%AB 7.2%A .oo

36 REFERMENTATION 38.5%B 16.4%A 44.3%B .00

ABC

Means not sharing same superscript are different (P < .01).

No change in relative proportions of specific fungi resulted
from the addition of organic acids. The primary effect of the acids
appears to be to delay fungal growth. Yeast play an important role

in the fermentation and refermentation of stored forages and are not

75

know to produce toxins during feed storage. Upon spoilage Aspergil-
lus became dominant but was not affected by treatment so upon spoil-
age the same possibility exists of toxin contamination in acid

treated silage as untreated but the time until spoilage is increased

and nutrient loss decreased by the acid treatments (particularly

propionic).

Summary and Conclusions

 

Organic acids can be used to decrease refermentation in high
moisture feeds. Pr0pionic appears to be much more effective than
formic acid in feed preservation. The addition of formic acid to
propionic acid decreased the effectiveness of propionic acid. Pro-
pionic acid significantly (P < .Ol) increased the number of days un-
til spoilage. The addition of organic acids did not change the type
of fungal flora present but delayed its growth. All levels of formic
acid severely inhibited lactic acid production while the inhibition
on the propionic treatment was small at the .5% treatment level and
became severe at 2%. At the present cost of acid it appears that
the maximum level of addition is approximately .5%. At this level
propionic acid appears to be a suitable additive to decrease spoilage

and refermentation.

 

Literature Cited

 

Albright, J. E., S. D. Aust, J. H. Byers, J. E. Fritz, B. 0. Brodie,
R. E. Olson, R. P. Link, J. Simon, H. E. Rhoades, and R. L.
Brewer. 1964. Moldy corn toxicosis in cattle. J. Amer. Vet.
Med. Assoc. l44zl0l3.

Barker, S. B. and w. H. Summerson. 1941. The colorimetric determina-
tion of lactic acid in biological material. J. B. C. 138:535.

Carpintero, M. C., A. J. Holding, and P. McDonald. l969. Fermentation
studies on lucerne. J. Sci. Fd. Agric. 20:677.

Castle, M. E., and J. N. Watson. 1970. Silage and milk production, a
comparison between wilted and unwilted grass silages with and
without formic acid. J. Brit. Grassld. Soc. 25(1):65.

Christensen, C. M. 1946. The quantitative determination of molds in
flour. Cereal Chem. 23:322.

Christensen, C. M., J. H. Olafson, and w. F. Geddes. T949. Grain
storage studies. VIII. Relation of molds in moist stored
cottonseed to increased production of carbon dioxide, fatty
acids and heat. Cereal Chem. 26:109.

Daniel, P., H. Honig, F. Weise, and E. Zimmer. l970. Mirkung von
pr0pionsaure bei der grunfuttersilierung. Das Wirtschaftseigene
Futter. 3:239.

Federson, E. J. N. l97l. Investigations on oxidation damage in silage.
Crop Sci. Res. Coun. Bull. (Denmark). 947:233.

Gilman, J. C., and D. H. Barron. 1930. Effect of molds on temperature
of stored grain. Plant Physiol. 5:565.

Henderson, A. R., P. McDonald, and M. K. Woolford. 1972. Chemical

changes and losses during the ensilage of wilted grass treated
with formic acid. J. Sci. Fd. Agric. 23:1079.

76

77

Honig, H. 1969. The influence of different anaerobic storage condi-
tions on fermentation. The 3rd Congress of European Forage and
Pasture Societies. Bull. 3:173.

Huber, J. T. 1970. Formic acid treatment of urea corn silage harvested
at different maturities. J. Animal Sci. 31:244.

Huber, J. T., R. E. Litchenwalner, D. D. Makdani, and H. E. Henderson.
l972. Influence of various organic acids on silage fermenta- .
tion. a. Animal Sci. 35 230 (abstr.). I]

Langston, C. W., H. Irvin, C. H. Gordon, C. Bouma, H. G. Wiseman, C. G.
Malin, and L. A. Moore. 1958. Microbiology and chemistry of
grass silage. U.S.D.A. Tech. Bul. 1187. r

 

Lynch, G. P., C. R. Benjamin, N. M. Jacoby, and C. M. Chance. l969. é?
Field report of high moisture, moldy corn tetany in milking
cows. J. Dairy Sci. 52:930.

Milner, M., and w. F. Geddes. 1945. Grain storage studies. II. The
effect of aeration, temperature and time on the respiration of
soybeans containing excessive moisture. Cereal Chem. 22:484.

Milner, M., and w. F. Geddes. l946. Grain storage studies. IV. Bio-
logical and chemical factors involved in the spontaneous heating
of soybeans. Cereal Chem. 23:449.

Mohanty, G. P., N. A. Jorgensen, R. M. Luther, and H. H. Voelker. 1969.
Effect of molded alfalfa hay on rumen activity, performance and
digestibility in dairy steers. J. Dairy Sci. 52:79.

Richardson, L. R. and J. V. Halick. l957. Studies on feed spoilage.
Texas Agr. Expt. Sta. Bull. 879.

Sleiman, F. T. l972. Effect of organic acid treatment on preserva-
tion, fermentation and nutritive value of unprotected forages
and high moisture ear corn. Ph.D. Thesis, Michigan State Uni—
versity.

Sleiman, F. T., J. T. Huber, and R. E. Litchenwalner. l972. Preserving
unprotected corn forage with organic acids. J. Animal Sci.
35:234 (abstr.).

Still, P. E., A. w. Macklin, w. E. Pibelin, and E. B. Smalley. l97l.
‘ Relationship of ochratoxin A to fetal death in laboratory and
domestic animals. Nature. 234:563.

78

Neise, F. l97l. Effect of propionic acid on growth of yeasts and molds.
Personal communication.

Zimmer, E., and C. H. Gordon. 1964. Effects of wilting, grinding, and
aerating on losses and quality in alfalfa silage. J. Dairy Sci.

47:652.

 

IV.
PART B: FUNGAL GROWTH DURING FERMENTATION
AND REFERMENTATION OF NON—PROTEIN-NITROGEN

TREATED CORN SILAGE

Abstract

Fifty-six kg portions of chopped whole corn plant (35% DM)
were treated with either urea, aqua-ammonia, or ammonia-molasses solu-
tion at 0%, .2%, .4%, or .8% added nitrogen, placed in polyethylene
bags inside 200 Q metal drums, evacuated, and sealed. During fermen-
tation forages were sampled and temperature measured on days 0, 5, 20,
and 86. On day 86 two 12 kg portions were placed in open containers
at 25 C. On days 0, 7, 21, and 28 of refermentation silages were
sampled, aerated and temperatures measured. Samples were analyzed for
VFA, lactic acid, pH, and number and type of fungi. Initial pH in-
creased with nitrogen addition. At .8% nitrogen pH values for urea,
aqua-ammonia, and ammonia-solution were 6.35, 9.88, and 9.65 respec-
tively. Added nitrogen also resulted in large pH increases during
refermentation. Ammonia added at .2% nitrogen stimulated, but higher
levels depressed lactate production in silages. Upon spoilage lactic
acid disappeared. Silages treated with the ammonia-solution required
the longest time to spoil (P < .01). Ammonia nitrogen reduced total
fungal counts (P < .01) at 30 minutes after treatment but during fer-
mentation no differences in fungal growth between treatments were

noted. At both .4 and .8% nitrogen fungal counts were lower (P < .01)

79

 

80

than at O and .2%. Treatment did not significantly change the rela-
tive proportions of fungi during spoilage. The majority were yeast

and Geotrichum, while some Aspergillus and Penicillium were identified.

 

 

 

However, yeast decreased and Geotrichum increased during Spoilage on

 

all treatments (P < .01).

 

 

Introduction U,“

Mohanty gt_al. (1969) fed moldy alfalfa hay to ruminants and
found reduced dry matter intake, body weight gains, total VFA, ruminal
ammonia, and total rumen protozoa. Albright ej;al, (1964) diagnosed
a gross hemorragic syndrome in 20 of 29 heifers fed a ration contain-
ing toxin-producing fungi. Fungal growth has been reported to cause
tetany in milk cows (Lynch gjgal, 1969); and Still gt_al, (1971) iso-
lated Aspergillus ochraceus from moldy hay which they reported as the
cause of abortions in dairy cattle.

Tomkins and Trout (1931) found green rot in citrus was largely
reduced by storage in a dilute ammonia atmosphere which could be pro-
vided by ammonium bicarbonate but not ammonium acetate. Ammonia in-
creased the latent period of fungal spore germination (Tomkins, 1932).

Bothast et a1. (1973) treated 12 or 26% moisture corn with ammonia at

81

2 or .5% of dry weight and reported that both concentrations elimin-
ated external and infecting molds and yeasts.

R Huber and Santana (1972) compared corn silage treated with an
ammonia solution at ensiling time with urea or control silages.
Heifers ate more ammonia silage than control and NPN supplemented
rations supported more milk production than the negative control.
Ammonia silage was higher in lactic acid, but there was no difference
in animal performance between the urea and ammonia treatments. How-
ever, more recent experiments suggest a slight superiority of ammonia
over urea.

The purpose of this study was to determine the effects of
urea, aquaeammonia and an ammonia-molasses-mineral mixture on fungal
growth and types of fungi during ensiling and refermentation of
chopped corn. In addition changes in fermentation acids, tempera-

ture, pH, and dry matter were determined.

Materials and Methods

Corn (approximately 35% dry matter) was field chopped and
brought to the storage area where duplicate fifty-six kg lots were
treated with O, .2, .4, or .8% added nitrogen as urea, aqua-ammonia

(aqua-NH4; water plus anhydrous ammonia, or an NH4-molasses, mineral

82

solution (NH -solution; trade name Prosil). This required the addi-

4
tion of .5, l,or 2% urea; l, 2, or 4% aqua-NH4; or 1.8, 3.6, or 7.2%
NH4-solution. The weighed material was spread on a polyethylene sheet
and sprinkled with the nitrogen compound. After blending with shovels
the entire mass was rotated on the polyethylene sheet to insure com- Fl
plete mixing. fl
Treated material was then transferred to 200 2 metal drums
lined with transparent polyethylene bags (5 mils in thickness). U
Samples (.5 kg) were removed and placed inside smaller bags and imme-
diately frozen at -20 C. The polyethylene bags containing the treated
material was then evacuated by the use of a vacuum cleaner and sealed.
Containers were stored in an enclosed barn on a concrete floor. On
days 0, l, 5, 10, 15, and 86 bags were opened, .5 kg samples were re-
moved quickly, and bags were re-evacuated and sealed. While the bags
were open temperatures were determined with mercury thermometers in-
serted into the center of the silage mass.
After 86 days of fermentation, 12 kg portions of the silage
were placed in open polyethylene containers and stored at 25 C. The
remaining silage was weighed and discarded. The material was aerated
by transferring from one container to another on days 1, 2, 3, 5, 7,

ll, 14, 21, and 27 and samples (.5 kg) were removed on days 1, 7, 21,

and 28.

83

Analyses of DM, pH, lactic acid, VFA, and fungi were performed

according to the procedures described in Part A.

Results and Discussion

The pH values for the ammonia treated silages were higher than
those for control or urea treatments 30 minutes after ensiling (Figure
4); but by 10 days pH of silages with .2% N as ammonia was similar to
control and urea treatments. Upon refermentation, the pH of nitrogen
treatments increased at a faster rate than controls. At the .8% N as
ammonia the pH during refermentation never declined below 6.0 and it
rapidly increased to 8.0 or more during refermentation (Table 1,
Appendix).

The pH values observed for ammonia added at .4 and .8% N were
above those for optimum growth of lactic acid bacteria (Langston gt
21, 1958); therefore decreased lactic acid was observed on these
treatments (Figure 5). In fact, despite an abundance of water soluble
carbohydrates lactic acid was almost completely inhibited at .8%
ammonia N, while less inhibition occurred at the .4%. At the .2%
ammonia N lactic acid was higher than controls or .2% urea N; whereas,
a stimulation of lactic acid was observed at both .4 and .8% urea N.

The pH of urea treated silages was never above 5 except early in

 

 

84

 

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86

fermentation and late in refermentation. (Complete data for lactic
acid production in Appendix, Table 2.)

Lactic acid production (Figure 6) on control and .2% urea N
was almost complete 10 days after ensiling while the higher peaks for
the ammonia treatments occurred on day 20. The ammonia buffered the
products of fermentation, thus extending the period of lactic acid
production. Lactic acid remained stable until refermentation at
which time concentrations decreased to negligible levels at spoilage
as reported by Sleiman (1972).

No differences in the maximum temperature during refermenta-
tion were observed between treatments or levels of nitrogen addition
(Table 13). This may have been due to the small mass of the silage
(12 kg) and the high constant temperature (25 C) of the storage room.
Small changes in silage temperature would have been overshadowed by
the room temperature. Neither were any differences seen in tempera-
ture during fermentation. Again large changes in ambient conditions
(21 C and 5 C) and the small mass of silage may have masked changes.
However, if complete anaerobic conditions were achieved little heat-
ing would have been expected (Federson, 1971).

Time until spoilage was observed (mold growth; Table 13) was
increased at both the .2 and .4% N additions so it appears that added
N as ammonia or urea (which partially degrades to ammonia) has some

value in preservation of forages. At the .8% N, spoilage was fastest,

87

 

 

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88

TABLE 13.--Maximum Temperatures, Days until Spoilage and Days until
Maximum Temperature During Refermentation as Affected by Different
Levels and Sources of N Addition to Corn Silage

 

 

MAXIMUM DAYS UNTIL DAYS UNTIL
TREATMENT TEMPERATURE MAXIMUM
(C) SPOILAGE TEMPERATURE
A
CONTROL 32.0 4.0 10.5
UREA 32.5 7.3AB 15.3
NH4-SOLUTION 33.0 12.4B 16.5
AQUA-NH4 33.4 4.6A 13.0
LEVEL OF
TREATMENT
0.0 32.2 4.0 14.5
0.2 32.2 11.3 17.8
0.4 35.5 11.5 16.8
0.8 32.0 4.0 10.5

 

ABMeans not sharing same

superscript are different (P < .05).

89

requiring only 4 days. Thus, the benefit of N in preservation is not
due to N per se but to changes mediated through fermentation. Days
until spoilage (Table 13) was increased (P < .05) by the NH4-solution
compared to control and aqua-NH4. Ammonia alone appears not to be as
effective a fungicide in preservation of forages as in high moisture
corn (Bothast §t_gl, 1973). However, all nitrogen treatments in-
creased days until spoilage. Days until maximum temperature closely
paralleled days until spoilage although no significant differences
were found. Maximum temperatures required approximately the same
amount of time after fungi were first detected regardless of the time
required for mold growth (spoilage) to begin.

Ammonia was very effective in destroying fungi present in
fresh-cut corn silage (Table 14) as previously reported by Bothast gt_
31, (l973) and Tomkins and Trout (1931). The effect was apparently
due to the NH4 ion and was not exhibited by urea. HoweVer, after
fermentation was initiated fungal growth was greatly decreased on all
treatments which explains why no significant differences in fungal
counts were seen between urea and ammonia during fermentation, which
coincides with anaerobic conditions being reached (Federson, 1971).

During fermentation both ammonia treatments had an increase
in fungal colonies which was larger on the higher treatment levels.
(Table 14 and Figure 7) This was due to the higher pH on these

treatments which inhibited lactic acid production and left sufficient

 

90

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92

oxygen and water soluble carbohydrates to permit fungal growth. How-
ever, the urea and low level of ammonia N had extensive fermentation
and fungal growth was comparable with controls. During ensiling
normal fermentation appears to be as efficient a fungicide as ammonia
or urea which is hydrolyzed to ammonia during fermentation.

Upon aeration or initiation of refermentation all silages
exhibited a rapid increase in fungal numbers but thereafter fungal
numbers for the ammonia-treated silages decreased at a faster rate
than for the control or urea silages (Table 14 and Figure 7). There
was a marked depression of fungi by all nitrogen treatments compared
to the control with a significant difference (P < .01) being seen at
the .4 and .8% N treatments; however, no treatment differences were
detected. A rapid decrease in fungal numbers was noted on the ammonia
treatments during refermentation due to the release of ammonia during
refermentation. This effect was more pronounced on the higher nitro-
gen treatments (Table l4) because of more ammonia release. Fungi on
the high urea treatments decreased rapidly during the later stages of
refermentation indicating extensive release of ammonia. The control
also exhibited a decrease in fungal numbers due to loss of lactic acid
and possibly deamination of protein and a decrease in available
nutrients.

Dry matter losses were 1.84, 2.55, 4.08, and l.43% respectively

for the 0.0, 0.2, 0.4, and 0.8% N treatments. The higher dry matter

 

93

losses at .2 and .4% N might have been due to a more extensive fermen-
tation indicated by higher lactic acid.

Yeast decreased (P < .Ol) as a per cent of total population
during fermentation and refermentation (Table l5) while Geotrichum

increased (P < .01). Penicillium were isolated only at the end of

 

fermentation and no significant numbers of Aspergillus were seen at

 

any time. Aspergillus were not detected in spoiled material and the
spoilage appeared to be due largely to bacteria because of the low
fungal p0pulation (Table l4) at the end of refermentation. Geotrichum
was the fungus which grew most at spoilage and was associated with
visual molding. No differences were observed between treatments in
type or relative proportions of fungi; hence, NPN has no apparent
effect on relative types but delays overall fungal growth.

TABLE lS.--Relative Proportions of Fungi in Corn Silage Treated with
Various Kinds of NPN

 

 

 

 

 

DATE YEAST GEOTRICHUM PENICILLIUM
DAY 0 90.0%A .3%A 0.0%B
EEEMEETATION 83.3%A 12.1%AB 4.5%A
EEEEgMENTATION 60-3%B 27-3%B 0.0%B

 

ABMeans in same column with different superscripts different (P < .01).

CInsignificant number of Aspergillus found.

 

94

Summary and Conclusions

Nitrogen Treatment decreased the number of total fungi. With
the exception of 30 minutes after addition no significant differences
between treatments were observed during fermentation. The addition of
more than .2% ammonia nitrogen severely inhibited lactic acid produc-
tion and was associated with higher fungal counts before opening the
silos. Ammonia increased the days until visual spoilage was detected,
but no significant changes between treatments were noted for fungal

types. Yeast decreased and Geotrichum increased during fermentation

 

and refermentation. Insignificant numbers of Aspergillus and Penicil-

 

ligm_were found.

These data indicate that in addition to supplying NPN, ammonia
can be used to inhibit fungal growth and increase stability of silage
when exposed to air. More than .2% nitrogen as ammonia depresses the

quality of silage as indicated by decreased lactic acid production.

’1'~. In

 

WISE..-

Literature Cited

 

Albright, J. L., S. D. Aust, J. H. Byers, T. E. Fritz, B. O. Brodie,
R. E. Olson, R. P. Link, J. Simon, H. E. Rhoades, and R. L.
Brewer. l964. Moldy corn toxicosis in cattle. J. Amer. Vet.
Med. Assoc. l44lel3.

Bothast, R. J., E. B. Lancaster, and C. N. Hesseltine. I973. Ammonia
kills spoilage molds in corn. J. Dairy Sci. 56:24l.

Federson, E. J. N. 1971. Investigations on oxidation damage in silage.
Crop Sci. Res. Con. Bul. (Denmark). 947:233.

Huber, J. T. and 0. P. Santana. l972. Ammonia treated corn silage for
dairy cattle. J. Dairy Sci. 55:489.

Langston, C. W., H. Irving, C. H. Gordon, C. Bouma, H. C. Wiseman,
C. G. Malin, and L. A. Moore. 1958. Microbiology and chem-
istry of grass silage. USDA Tech. Bul. ll87.

Lynch, G. P., C. R. Benjamin, N. M. Jacoby, and C. M. Chance. l969.
Field report of high moisture moldy corn tetany in milking
cows. J. Dairy Sci. 52:930.

Mohanty, G. P., N. A. Jorgensen, R. M. Luther, and H. H. Voelker. 1969.
Effect of molded alfalfa hay on rumen activity, performance, and
digestibility in dairy steers. J. Dairy Sci. 52:79.

Sleiman, F. T. l972. Effect of organic acid treatment on preserva-
tion, fermentation, and nutritive value of unprotected forages
and high moisture ear corn. Ph.D. Thesis, Michigan State Uni-
versity.

Still, P. E., A. w. Macklin, w. E. Pibelin, and E. B. Smalley. l97l.

Relationship of ochratoxin A to fetal death in laboratory and
domestic animals. Nature. 234:563.

95

 

96

Tomkins, R. G. l932. The action of certain volatile substances and
gases on the growth of mould fungi. Proc. Roy. Soc. (London).
lllB:210.

Tomkins, R. B. and S. A. Trout. l93l. The use of ammonia and ammonium
salts for the prevention of green mould in citrus. J. Hort.
Sci. 9:257.

 

‘W -

V.
PART C: PRESERVATION AND ANIMAL PERFORMANCE OF
HIGH MOISTURE CORN TREATED WITH
NON-PROTEIN-NITROGEN AND

PROPIONIC ACID

 

Abstract

High moisture shelled corn harvested at approximately 27%

 

moisture was treated with: l) propionic acid (l.2%), 2) a mixture ??
of 80% propionic and 20% acetic acid (l.2%), 3) aqua-ammonia (at a

.54% NH3) or 4) a commercial ammonia-solution (at .63% NH3). For .
treatments 1, 3, and 4 approximately l50 bushels of corn was treated E;

and stored in gravity wagons until feeding. Only 5 bushels of corn
for laboratory use was treated with the propioniczacetic mixture.
Recovery of nitrogen for the ammonia treatments was 60% for
the ammonia-solution and 30-40% for the aqua-ammonia while 80% of the
propionic was recovered. No significant changes in protein or acid
content were observed during storage. Fungi were reduced by all addi-
tives (Zl vs 690 colonies/g X 103) 30 minutes after treatment. Fungal
counts after 28 days for the aqua-ammonia and control treatments were
significantly higher than the others. During late storage both ammonia
treatments showed increases in fungal colonies. These occurred earlier
and were of greater magnitude for the aqua-ammonia than on the ammonia-
solution. After 60 days of storage corn treated with aqua-ammonia
heated to 50 C while the other treatments remained at ambient tempera-
ture. After rolling the corn prior to feeding the aqua-ammonia corn

97

98

had higher fungi (P < .05) than other treatments. After rolling in a
hammer mill, corn treated with the ammonia-solution heated to 30 C
while the propionic remained at ambient temperature (4-10 C) and had
not heated 60 days after rolling.

Lactating dairy cows (8 per group) readily consumed all corns
in a 5-week feeding trial, even though that treated with aqua-ammonia
was heavily molded. No health problems were detected in any of the
cows due to eating the moldy corn. Milk production was 21.2, 18.4 and
19.3 kg for treatments l, 3, and 4 respectively (P < .109) and persis-
tencies of milk yield (trt/std) averaged 93, 88, and 81% for the re-

spective treatments (P < .l26).

Introduction

 

Changes in harvesting methods and difficulty in drying wet
corn has created increased interest in high moisture (HMC). The
storage of HMC has caused problems of molding. Trenk and Hartman
(1968) concluded that at moistures above 17.5% and temperatures
greater than 13 C corn was susceptible to aflatoxin formation by

Aspergillus flavus. Lynch et a1. (1969) found a 6% decline in milk

 

production when hay molded with Aspergillus and Penicillium was fed

 

 

to cows and Mirocha et a1. (1968) reported that feeding moldy hay to

 

 

99

150 dairy cows increased the number of services per conception from
1.2 to 4.0.

Preservation of HMC with organic acids is now competitive with
drying. Porter (1946) found organic acids had a greater microbiolog—
ical inhibitory effect at the same pH than mineral acids which was
attributed to the whole molecule. Richardson and Halick (1957) found
propionic acid was fungicidal in corn meal at .1%, while .3% calcium
propionate was required for equal protection and sodium propionate
was not effective at .6%. Propionic acid prevented mold growth in HMC
(Jones, 1970; Arends §t_al, 1970).

An even less expensive method of preserving HMC was reported
by Bothast gt_al. (1973). They treated 12% moisture and shelled corn
reconstituted to 26% with ammonia at 2 or .5% of dry weight. Both
concentrations of ammonia eliminated external and infecting molds and
yeasts.

The objective of this study was to compare ammonia, and pro-
pionic acid as a fungicide for HMC. The nutritive values of treated

grains for lactating dairy cows was also ascertained.

 

100

Materials and Methods

 

TRIAL I: High moisture shelled corn (HMC) containing approxi-
mately 27% moisture was harvested in January, 1973 and treated with
propionic acid (1.2%) or an 80% propionic, 20% acetic mixture (1.2%)
called chemstor.A Acids were sprayed onto corn as it passed through
an auger designed for that purpose.A Two additional lots of corn were
treated with aqua-ammonia (aqua-NH4; 22%N) applied at .54% NH3, or an

ammonia-molasses-minerals mixtureB (13% N; NH -solution) applied at

4
.63% NH3. The ammonia materials were added to the corn as it entered
a forage blower after metering through a calibrated pump. Approxi-

mately 150 bushels of corn were treated with propionic, aqua-NH4, or

the NH -solution and augered into gravity wagons until fed. Only

4
about 5 bushels was treated with chemstor which was sufficient for
laboratory tests.

From all treatments 35 kg portions of HMC were placed in open
polyethylene containers and stored at 25 C. Temperature VFA, lactic
acid, protein, DM, pH, and fungal counts and identification were

measured on samples collected on days 0, 2, 5, 9, 16, 23, 30, and 37

of storage. Samples were stored at -20 C until analyzed. After 90

 

AFurnished by Celanese Chemical Co., Corpus Christi, Texas.

BFurnished by Ruminant Nitrogen Co., Adrian, Michigan (Trade
name--Prosi1).

 

 

101

days of storage in the wagons, additional 35 kg portions were placed in
a 25 C room as previously described and sampled at days 0, 3, 7, 10,
17, and 31.

Rolling HMC before feeding is generally recommended. The HMC
was removed from the storage wagons and passed through a roller mill
in preparation for feeding to dairy cows. Rolled corn was placed in
200 z barrels and stored in a barn at ambient temperatures. Samples
were collected on days 0, 4, 6, 9, l2, and 13 and analyzed as previ-
ously outlined.

Corn samples remained frozen at -20 C until analyzed. For DM
determination corn was ground and 20 g were placed in a forced air
oven at 90-100 C for 24 hours. A homogenate for pH, lactic acid, VFA,
and fungal analyses was prepared by blending a 40 g aliquot of HMC
with 160 m1 of distilled sterile water for 3 minutes in an omni-mixerA
with the cup immersed in ice. Approximately 50 m1 of the homogenate
was placed in a separate sterile container for plating of fungal popu-
lations. The pH was measured with a Sargent pH meter.B Extracts were
prepared by straining the homogenate through two layers of cheesecloth.
Extracts were deproteinized with 50% SSA and analyzed for lactic acid,

VFA, and fungi as described in Part A for corn silage.

 

AIvan Sorvall Inc., Newton, Connecticut.

BE. H. Sargent and Co., Chicago, Illinois.

 

 

102

TRIAL II: The rolled HMC from the storage wagons in Trial I
was fed for 5 weeks to 24 lactating dairy cows averaging between 14
and 36 kg of milk per day. Cows were blocked according to production
during pre-treatment and randomly assigned to the three HMC feeds.
The HMC was fed at 1 kg per 2.5 kg of milk produced during pre-
treatment, corn silages at 11.4 kg per day and alfalfa haylage ad
lib. During the experimental period corn silage, haylage, and HMC
were sampled 3 times weekly. Samples were composited on a weekly
basis and frozen for analyses. Orts were measured daily and daily
feed, DM, and protein intake were determined. Milk production and

butterfat percent were calculated on a weekly basis.

Results and Discussion

 

Trial 1: The acids caused no visual change in the color of
corn kernels while both ammonia treatments darkened the seeds imme-
diately after application. It was planned to apply the ammonia at
equal rates but due to difficulties in the calibration of the pump

the NH -solution was applied at .63% NH (.85% of DM) and the aqua-

4

NH4 at .54% NH3 (

addition of .5% NH4 (of dry weight) to 27% moisture corn destroyed

3
.73% of DM). Bothast et al. (1973) reported that

 

103

both external and infecting fungi. Hence, the level of ammonia in my
study should have prevented fungal growth.

Organic acid levels (1.2%) were within the range which inhib-
ited fungal growth in 27% moisture corn (Miller, 1971). The amount
of propionic acid needed for preservation was directly proportional

to the moisture content of the HMC.

Temperatures of grain in the wagons were checked periodically.

After 60 days of storage, temperatures of the aqua-NH4 corn increased
above the ambient (7-14 C) and reached 40-50 C within 10 days; while
temperatures of corn treated with propionic and NH4-solution remained
at about ambient. Heating forced the feeding trial to be initiated
earlier than had been planned. These data show that the aqua-NH4 was
ineffective in preventing heating in stored HMC for 60 days, although
it was effective for approximately 30 days during which time control
corn became heavily molded. Bothast §t_al. (1973) found ammonia
treatment preserved corn for 14-21 days. Propionic acid and the NH4-
solution prevented heating for the entire period the HMC was stored
prior to feeding.

To test the preservative effects of NPN and organic acids
under better controlled storage than gravity wagons, 35 kg portions
were placed in polyethylene containers and stored at 25 C. In these

samples no lactic acid was detected at any date. A lack of fermenta-

tion was also implied by the pH values (Table 16), which did not

 

 

104

TABLE l6.--The pH During Storage of HMC Treated with Organic Acids

or NPN

 

Days of Storage

 

 

 

 

2 9 23 30
PROPIONICA 4.50 4.50 4.50 4.50
CHEMSTORA 4.55 4.55 4.55 4.60
AQUA-NH4B 7.85 7.35 6.70 6.70
NH4-SOLC 9.00 8.45 8.35 8.35
CONTROLD 6.20 6.45 6.20 6.35
ABCD . . . . . .
Treatments not sharing same superscrIpts s19n1f1cantly d1fferent
(P < .01).

change during storage. The acid additions significantly decreased pH
(P < .01) compared to untreated corn; while the ammonia increased pH,

with the NH -solution higher (P < .01) than the aqua-NH due to a

4 4

higher level of application.

The nitrogen content of the HMC was increased more by the NH4-
solution than the aqua-NH4 (Table 17) due to the higher rate of appli-
cation and greater apparent loss of aqua during treatment (66 vs 40%).
The high apparent loss of nitrogen may have been due to miscalculated
flow rates and corn weight: Hence, it is impossible to accurately de-

termine the true loss of ammonia during treatment. There was no

105

TABLE l7.--Crude Protein (% of DM) of HMC During Storage Treated with
NPN and Organic Acids

 

Days of Storage

 

 

 

2D 9D 23DE 30E
PROPIONICA 10.25 9.89 10.25 10.34
CHEMSTORA 9.79 9.56 10.44 10.07

18
AQUA-NH4 11.27(34) ll.ll(26) 11.44(39) 11.39(57)
1c

NH4-SOL 12.06(48) 12.50(58) 12.63(61) 13.16(73)
00NTR0LA 9.90 10.11 9.90 10.25
ABC

Treatments not sharing the same superscripts are significantly
different (P < .01).

DEMeans not sharing the same superscripts significantly different
(P < .01).

1Percent recovery of nitrogen or the NH
parenthesis.

4 treatments given in

 

106

apparent loss of nitrogen (ammonia) during storage. In fact, after 30
days of storage protein was significantly higher on all treatments
than at 2 days. After adding to HMC, ammonia is tightly bound, so the
poor initial recoveries are not due to losses after treatment. Some
losses did occur during treatment because of the presence of an ammonia
odor in the area of treatment; however, the accurate determination of
the extent of these losses cannot be determined from these data.
Estimated recoveries of propionic acid (Table 18) averaged 8%
on the propionic treatment and 82% for chemstor. Five of eight de-
terminations indicated almost total recovery and differences in indi-
vidual sampling may explain the low estimates (41 and 63%) obtained
in two samples.

TABLE 18.--Propionic Acid (% wet wt) of HMC Treated with NPN and
Organic Acids After Storage

 

J J
1.. ~— W—

Days of Storage

ll

 

 

 

3 9 23 3O
PROPIONIC1 1.25(100) .76(63) 1.17(98) 1.11(93)
CHEMSTOR1 .39(41) 1.12(117) .91(95) .72(75)
AQUA-NH4 .02 .06 .02 .08
NH4-SOL .06 .11 .08 .01
CONTROL .02 .08 .02 .06

 

 

1 . . . . .
Percent recovery of prop10n1c 91ven 1n parentheSTS.

107

Total fungi were significantly depressed by all treatments
(Figure 8; complete data in Appendix, Table 3). Thus organic acids
and ammonia were equally effective in destroying initial fungi. By
7 days fungal counts in both ammonia treatments began to increase,
but at 28 days, corn treated with the NH4-solution reverted back to

counts similar to those for the acid treatments; while those for

aqua-NH continued to increase. Corresponding to the higher fungal

4

counts in the warm room; the aqua-NH corn was the first to heat in

4
the gravity wagons, exposed tocxflder ambient temperatures. Gilman and
Barron (1930) found that heating of grains was due primarily to fungal
growth.

The decreased mold inhibition on aqua-NH4 may have been due to

loss of the NH ion from the internal atmosphere of the corn because

4
of a possible linkage with carbohydrates. Tomkins and Trout (1931)
reported that 500-1000 PPM of ammonia in the atmosphere was required
to prevent fungal growth. Loss of the ammonia odor from the grain
occurred about the time heating and fungal growth began. Bothast §3_
.11. (1973) reported that ammonia killed initial fungi in HMC as in
this data but after ammonia disappeared from the atmosphere surround-
ing the HMC, spores were still present to initiate new fungal growth
which was largely uninhibited (Christensen and Gordon, 1948). Thus,

ammonia appears to be a very effective short-term grain preservative.

Its value is rapidly lost during storage under minimal protection.

 

 

108

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109

Another possible explanation for the poorer fungicidal action of
aqua-NH4 was the low ammonia level (.31% of the corn BM) in this

treatment, whereas the NH -solution contained (.51% of DM) similar

4

to that shown to have fungistatic properties by Bothast et al. (1973).

Relative proportions of fungi did not change and spoilage was

largely due to yeast and Geotrichum; however, some Aspergillus and

 

Penicillium were observed. The effectiveness of propionic acid in

 

preventing fungal growth agrees with Porter (1946), Richardson and
Halick (1957), Weise (1971), and Jones (1971). There were numerous
fungi present at harvest since the corn had been standing in the
field ready to harvest for 90 days. The propionic was fungicidal
to this population in support of Singh-Verma (1971) who found the
acid most effective after deterioration had begun because cells were
growing in their log phase.

The next two sections of Trial I are very similar to the pre-
vious section and the data will only be discussed as it contradicts
data from the first section. The second section compared aqua-NH4,

NH -solution, and propionic acid treatments during refermentation.

4
This was corn which was treated and stored as described in section 1
and removed from the gravity wagons (after 60 days) at initiation of
this experiment.

The pH of propionic-treated HMC remained constant after re-

moval from the gravity wagons throughout the entire period (Table 19)

 

 

110

TABLE l9.--PH and Protein1 (% of DM) of HMC Treated with NPN and
Organic Acid During Refermentation

 

Days of Refermentation

 

0D 3DE 7DE 10E 17E

 

CRUDE PROTEIN

AQUA-NH41 11.44(39) 11.43(39) 11.42(39) 11.92(52) 11.99(54)

 

 

 

NH4-50L18 12.44(56) 12.61(60) 12 12(49) 12.53(59) 12 53(59)
PROPIONICC 10.16 10.17 10.14 10.31 10.21

PH

AQUA-NH4A 7.90 7.67 7.40 7.23 6.80
NH4—SOLB 8.40 8.28 7.82 7.50 7.85
PROPIONICC 4.65 4.65 4.65 4.58 4.67
ABC

Treatments not sharing same superscripts different (P < .01).
DEDates not sharing same superscripts different (P < .05).

1Nitrogen recoveries (%) in parentheses.

 

111

while that of ammonia treatments significantly decreased (P < .05)
during refermentation indicating production of acids or loss of the
additive. The ammonia had higher pH values than the propionic (P <
4 was lower (P < .01) than the NH4-solution.

Crude protein was also higher (P < .01) for the ammonia treatments

.01) and the aqua-NH

with the NH4-solution higher than aqua. Nitrogen recoveries averaged
56.6% for the NH4-solution and 44.6 for aqua. No apparent losses
occurred over storage so the decreased pH was probably due to acid
production. Neither was there loss of propionic acid after 3 days

of storage (Table 20); however, there appears to be a loss between

days 0 and 3 of storage which may have been due to sampling error.

TABLE 20.--Propionic Acid (% wet wt) of HMC Treated with NPN and
Organic Acids During Refermentation

 

 

Days of Refermentation

 

 

O 3 7 1O 17
AQUA-NH4 .07 .05 .05 .06 .06
NH -SOL .16 .08 .09 .08 .03

4

PROPIONIC1 1.26(1.05) .90(75) .80(67) .88(73) .89(74)

 

l . . . . .
PropionTc ac1d recoveries (%) 1n parentheses.

112

Fungal data is given in the Appendix (Table 4). Fungal growth

(Figure 9) began to increase in 3 days on the aqua-NH and at 10 days

4
on the NH4-solution while remaining at near zero on the propionic
treatment. At 31 days the ammonia treatments had significantly more

(P < .10) fungi than the propionic. Geotrichum and yeast were again

 

responsible for spoilage, while some Aspergillus and Penicillium were
detected. No differences in temperatures were observed.

In section 3 the corn was rolled and 120 kg portions were
placed in steel drums for storage prior to feeding. Because of a
mechanical problem all the propionic-treated HMC had to be rolled
at the initiation of the trial but no heating or fungal growth was
evident during the 40 days of feeding while the ammonia-treated HMC
was rolled twice weekly. The pH values for the propionic treatment
(Table 21) remained stable throughout storage while the ammonia treat-
ments decreased, reflecting loss of ammonia or formation of an ammonia-
carbohydrate complex. The protein content remained stable throughout
storage so formation of a complex appears unlikely. The NH4-solution
treated corn had higher pH and protein than the aqua (P < .01). Pro-
pionic acid recoveries after rolling were much higher than in either
of the two previous studies averaging 167% (Table 22). The only ex-
planation is that rolling stimulated propionate fermentation which

is not likely.

 

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113

 

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114

 

 

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116

The aqua-NH4 corn which had heated in the wagons was signifi-
cantly higher in fungi (P < .01) after rolling and throughout storage
(Figure 10). Fungi in the NH4-solution increased when compared to
propionic but no significant differences were seen. Fungal data is
in Appendix (Table 5).

The temperature (Figure 11) of the rolled HMC treated with
propionic acid remained close to ambient while the temperature of the
aqua-NH4 which had peaked in the wagons decreased. After three days
the temperature of the NH4-solution HMC increased until day 7 then
began to decrease. This heating closely parallels the increase ob-
served in fungal counts during the storage period and confirming the
role of fungi in grain heating. No differences in type of fungi were
noted between treatments.

Trial II: As indicated by storage data, heating of the aqua-
NH4 treated HMC necessitated beginning feeding earlier than antici-
pated. Because of this, cows had not been on a standardization ration
so they were allotted to treatment according to milk yields for the 28
days before this trial started. No fat tests were available during
the period so it was not possible to co-vary fat corrected milk (FCM)
during the trial and pre-treatment values as is usually done.

Milk production (Table 23) adjusted for pre-treatment differ-
ences was depressed on aqua-NH4 (P < .109). There was also a decline

in fat percent of cows on aqua—NH4. Adjustment of this data to FCM

 

 

117

 

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119

TABLE 23.--Mi1k Production Data of Cows Fed HMC Treated with Either
Aqua Ammonia, Propionic Acid, or Ammonia Solution

 

 

 

TREATMENT
PROPIONIC AMMONIA AQUA LEVEL OF
ACID SOLUTION AMMONIA SIGNIFICANCE
MILK (COV. PRETRT.;kg) 21.2 19.3 18.4 .109
FAT (%) 3.56 3.53 3.37 .654
PERSISTENCY (PRETRT;%) 93.3 88.3 81.1 .126

 

would have probably shown a significant depression in FCM yield on
aqua-NH4. There was also a decline in milk yield persistencies on
the aqua-NH4 which approached significance (P < .126).

No intake (Table 24) or health problems were noted in animals
fed corn treated with ammonia, despite the extensive molding which had
occurred. Intake of corn DM averaged 7.0, 6.6, and 6.6 kg/day for
acid, NH4-solution and aqua-NH4 treatments, respectively. HMC treated
with ammonia had a faint ammonia odor and smelled moldy while the pro-
pionic had an acid smell, which apparently was not objectionable to
the cows. Neither were total DM intakes different between groups, but
protein intake was higher on the ammonia than the other treatment, be-
cause of the added nitrogen. Body weight gains were higher also on

the ammonia treatments. Christensen et a1. (1973) also reported no

 

 

i- - -
‘33—}: ~
I"

 

 

120

TABLE 24.--Body Weight Gain and Feed Consumption of Cows Fed Propionic
Acid, Aqua Ammonia or Ammonia Solution Treated HMC*

 

 

 

PROPIONIC AMMONIA AQUA
ACID SOLUTION AMMONIA
BODY NT GAIN (kg) 14.2 26.0 24.5 E,
HMC INTAKE (kg DM) 7.0 6.6 6.6 51 .
TOTAL 0M INTAKE (kg) 16.3 16.5 16.3
TOTAL PROTEIN INTAKE (kg) 1.93 2.16 2.10 1. u
1:913

 

*No means were significantly different (P < .10).

harmful effects on animals after feeding grain which was heavily in-

fested with a toxin-producing strain of Aspergillus flavus.

 

Summary and Conclusions

 

Ammonia treatment of HMC will reduce fungi; however, regrowth

 

of fungi may occur. The lower apparent recoveries of ammonia from
aqua-NH4 (30-40%)than from the NH4-solution (60%) were probably respon-
sible for the aqua treatment spoiling earlier. Propionic acid was a
very effective preservative and approximately 85% of that added was

recovered after storage at 25 C for 30 days. No apparent toxicity

121

was observed after feeding the moldy HMC to lactating dairy cows. The
decrease in milk production on the aqua-NH4 treatment approached sig-

nificance. Ammonia does not appear to be as effective a preservative

as propionic acid for HMC stored under minimal protection. However,

corn retaining at least .5% NH did not heat or spoil in gravity

3

wagons for about 100 days after harvest, while that with a lesser

ammonia concentration and control corn spoiled much more rapidly.

 

 

Literature Cited

 

Arends, L. G., R. D. Hyatt, M. H. Gehle, and P. B. Hamilton. 1972.
Preservation of high moisture corn with volatile fatty acids.
615t. Ann. Meeting Poultry Sci. Assn. P. 91. (abstr.).

Bothast, R. J., E. B. Lancaster, and C. N. Hesseltine. 1973. Ammonia
kills spoilage molds in corn. J. Dairy Sci. 56:241.

Christensen, C. M. 1973. Tests with propionic and acetic acids as
gain preservatives. Feedstuffs. FEB. 26:36.

Christensen, C. M., and D. R. Gordon. 1948. The mold flora of wheat
and corn and its relation to heating of moist grain. Cereal
Chem. 25:41.

Gilman, J. C. and D. H. Barron. 1930. Effect of molds on temperature
of stored grain. Plant Physiol. 5:565.

Jones, G. M. 1970. Preservation of high moisture corn with volatile
fatty acids. Can. J. Animal Sci. 50:739.

Jones, G. M. 1971. Utilization of volatile fatty acids in high mois-
ture grain preservative and dairy rations. Can. Res. Bu1. l.

Lynch, G. P., C. R. Benjamin, N. M. Jacoby, and C. M. Chance. 1969.
Field report of high moisture moldy corn tetany in milking
cows. J. Dairy Sci. 52:930.

Miller, J. I. 1971. Organic acid preservation of high moisture corn.
Feedstuffs. 43(25):20.

Mirocha, C. J., J. Harrison, A. A. Nochols, and M. McClintock. 1968.
Detection of a fungal estrogen (F-2) in hay associated with
infertility in dairy cattle. Appl. Microbiol. 16:797.

Porter, J. R. 1946. Bacterial chemistry and physiology. John Wiley
and Sons, Inc. New York, N.Y.

122

 

 

 

 

123

Richardson, L. R. and J. V. Halick. 1957. Studies on feed spoilage.
Texas Agr. Expt. Sta. Bull. 879:6P.

Singh-Verma, S. B. 1971. Einsatz der pr0pionsaure zur nachkonservierung
von getreide und kornermar's soure ihr wukung emechanismus.
Landwirtsch. Forsch Sonderheft. 26:71.

Trenk, H. L. and P. A. Hartman. 1968. Aflatoxin in stored corn. Bac—
terial. Proc. P. 2.

Tompkins, R. G. and S. A. Trout. 1931. The use of amnonia and amnonium
salts for the prevention of green mould in citrus. J. Hort.
Sci. 9:257.

Neise, F. 1971. Effect of propionic acid on growth of yeasts and molds.
Personal communication.

 

 

 

 

VI

SUMMARY AND CONCLUSIONS

Experiments were conducted with corn silage and high moisture
shelled corn to evaluate the preservative value of organic acids and
NPN additives on the fungal growth during fermentation and refermenta-
tion of the respective feeds.

Organic acids were effective in decreasing refermentation in
high moisture feeds. Pr0pionic appears much more effective than
formic acid in feed preservation. The addition of formic acid to
propionic decreased the effectiveness of the propionic. Propionic
acid significantly (P < .01) increased the number of days until spoil-
age. The addition of organic acids did not change the type of fungal
flora present but delayed its growth. All levels of formic acid
severely inhibited lactic acid production while the inhibition on
the propionic treatment was small at the .5% treatment level but be-
came severe at 2%. At the present cost of acid it appears to be a
suitable additive for decreasing spoilage and refermentation.

Nitrogen treatment decreased the number of total fungi. With
the exception of 30 minutes after addition no significant differences
between treatments were observed during fermentation. The addition

124

 

 

 

 

 

125

of more than .2% ammonia nitrogen severely inhibited lactic acid pro-
duction and was associated with higher fungal counts before opening

the silos. Ammonia increased the days until visual spoilage was de-
tected, but no significant changes between treatments were noted for

fungi types. Yeast and Geotrichum increased during fermentation and

 

 

 

 

refermentation. Insignificant numbers of Aspergillus and Penicillium F71
.
were found. 1
These data indicate that in addition to supplying NPN, ammonia _.«

is effective in inhibiting fungal growth and increases the stability L;

of silage when aerated. More than .2% nitrogen as ammonia depresses
the quality of silage as indicated by decreased lactic acid produc-
tion.

Recovery of nitrogen on the ammonia treatments of the high
moisture corn averaged 60% for the ammonia-solution and 36% for the
aqua-ammonia while 80% of the propionic acid was recovered. Fungi

were reduced by all additives 30 minutes after treatment. After 28

 

days fungal colony counts for the aqua-ammonia and control treatments
were higher than others. During late storage both ammonia treatments
increased in fungal populations but they occurred earlier and were of
greater magnitude on aqua-ammonia. After 60 days of storage aqua-

ammonia treated corn heated to 50 C. Upon rolling, corn treated with
the ammonia-solution heated while propionic corn remained at ambient

temperatures. Lactating dairy cows consumed all corn readily, even

126

though the ammonia-treated corn was molded, and no health problems
were noted. Milk production and persistency were less (P < .109) on
the aqua-ammonia than other treatments.

Although ammonia increased the stability of corn silage during
refermentation, it appears unsatisfactory as a long-time preservative
for high moisture corn stored under minimal protection. Propionic
acid treatment appears very satisfactory for preserving HMC and no
heating or fungal growth occurred during storage and feeding. No
acute mycotoxin poisoning of ruminant animals after eating heavily

molded corn were noted.

 

 

 

 

 

APPENDIX

 

APPENDIX

 

 

 

 

 

 

 

TABLE l.—-PH'sA in NPN Treated Corn Silage During Fermentation and
Refermentation.

Days of Days of 1.,

Fermentation Refermentation 4

O 5 20 86:0 7 21 29 '

Control 5.73 4.13 4.20 4.13 5.90 5.03 5.13 i} '
.5% Urea 6.25 4.35 4.48 4.35 4.70 6.85 7.93
1.0% Urea 6.95 4.73 4.63 4.50 4.65 6.83 6.78
2.0% Urea 6.35 4.98 4.90 4.53 4.58 6.78 7.33
1% Aqua-NH4 8.68 4.85 4.48 4.33 5.88 6.78 7.38
2% Aqua-NH4 9.38 6.85 5.30 4.88 5.40 6.43 7.53
4% Aqua-NH4 9.88 9.30 7.75 6.60 8.13 8.18 7.08
1.8% NH4-Sol 8.40 4.55 4.38 4.35 6.40 6.18 6.45
3.6% NH4-Sol 9.08 6.95 5.05 5.13 5.03 5.88 6.15
7.2% NH4-Sol 9.65 8.70 6.87 6.63 7.23 8.60 7.40

 

AEach value is the average of two duplicates.

127

 

APPENDIX

Table 2.--Lactic Acid ProductionA (% of BM) in NPN Treated Corn Silage
During Fermentation and Refermentation.

 

 

 

 

 

Days of Days of
Fermentation Refermentation
0 5 20 86:0 7 21 28
Control 0.22 4.06 5.61 5.81 2.25 2.78 0.84
.5% Urea 0.16 5.74 5.16 5.69 2.97 2.35 0.50
1.0% Urea 0.06 4.71 6.44 5.45 5.12 3.05 0.95
2.0% Urea 0.15 5.05 7.12 7.74 8.63 4.09 1.01
1% Aqua-NH4 0.16 4.62 7.20 7.10 2.41 2.37 0.50
2% Aqua-NH4 0.19 0.97 3.61 4.28 4.47 2.01 0.85
4% Aqua-NH4 0.06 0.00 0.90 0.65 0.63 0.60 0.45
1.8% NH4—Sol 0.43 4.64 9.21 5.94 3.14 2.20 0.75
3.6% NH4-Sol 0.12 0.46 5.77 4.61 3.55 1.17 0.86
7.2% NH4-Sol 0.03 0.00 0.29 0.45 0.39 0.30 0.43

 

AEach value is the average of two duplicates.

128

 

“1:.

4‘ 1L.—.___._~.1‘b

 

 

 

 

APPENDIX

TABLE 3.--Total Fungal Colonies (per g x 103) of HMC Treated with
Ammonia and Organic Acids

 

 

 

 

2 9 23 30
g-_
PROPIONIC 23 11 7 25
CHEMSTOR 5 4 6 13
NH4-SOLUTION 11 46 310 41
CONTROL 690 3,800 6,000 11,800
AQUA-NH 450 110 390 1,005

4

 

 

129

 

APPENDIX

TABLE 4.--Tota1 Number of Platable Fungal Colonies (X 103 per g) in
HMC Treated with Various Preservatives During Refermentation

 

 

 

 

 

11
”1
Days of Storage i
0 3 7 10 17
AQUA-NH4 3 94 415 22,250 32,250
NH4-SOLUTI0N 30 43 32 273 6,400
PROPIONIC 21 11 16 8 46

 

 

130

APPENDIX

TABLE 5.--Tota1 Fungal Colonies (per g x 103) in HMC Treated with
Ammonia and Propionic Acid After Rolling

 

Days After Rolling

 

0 4 6 9 12 13

 

AQUA-NH4 1,060 4,750 4,050 9,150 5,000 8,150
NH4—SOLUTION 123 35 106 124 210 52
PROPIONIC ACID 69 8 3 46 29 8

 

131

 

 

 

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