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31293 00901 1580

This is to certify that the

thesis entitled

EFFECTS OF MIA, SULFUR, MD
ISOACIDS ON _I_! VITRO RUHINAL
FERHENTATION 0F TROPICAL FORAGES

presented by
50L AMBEL RODRIGUEZ-MEDINA

has been accepted towards fulfillment
of the requirements for

 

 

Master of Sciencgegree in Animal Science

/€C”(M/7~ 679/4

Major professor

Date 2- 27-92

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

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PLACE IN RETURN BOX to remove this checkout from your record.
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iflfi _ 7 implant

 

 

EFFECTS OF AMMONIA, SULFUR AND ISOACIDS
ON IN VIIRO RUMIN AL FERMENTATION OF

TROPICAL FORAGES

By

Sol Anabel Rodriguez-Medina

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

MASTER OF SCIENCE

Department of Animal Science

1991

ABSTRACT

EFFECTS OF ANIMONIA, SULFUR AND ISOACIDS
ON IN VIIRO RUMINAL FERMENTATION OF
' TROPICAL FORAGES

By

801 Anabel Rodriguez-Medina

The effects of two levels of NH3, sulfur and isoacids on in vitro ruminal true
digestibility (IVTD) of eight tropical forages was studied using a 23 factorial design.
Trypticase was added to the final incubation media. This casein hydrolyzate should not
have been added because it contributes NH3, isoacids and sulfur. This raised the levels
of NH3 from theexpected 5-10 to 17-26 mg/dl and isoacid levels about 3-fold. Sulfur
levels were not increased. The results are discussed in light of trypticase addition. After
a 48 h in vitro ruminal fermentation, NDF, ADF and lignin were measured to estimate
IVTD. The results showed that IVTD increased with high levels of NH, and when
isoacids were added to treatments low in NH,. IVTD decreased when both NH; and
isoacids were high. High levels of sulfur tended to decrease IVTD for all forages,

except elephant grass. Addition of isoacids increased total VFA concentration.

I would like to dedicate this work to my beloved daughter, Aniella sanchez, who
will later understand the many sacrifices, patience, understanding, encouragement,
endurance and love behind this two long years for both of us. I hope it will serve her

as an example to achieve all her future goals in life.

ACKNOWLEDGENIENTS

I would like to thank my advisor, Dr. Robert Cook for his support, guidance,
sharing of knowledge and encouragement during the course of my graduate work as well
as his understanding and advice in difficult personal situations. A special thanks goes
to Dr. Telmo Oleas for his concern, invaluable advice, time, patience and his friendship.

I am grateful to Dr. Warner Bergen, Dr. Margaret Benson and Dr. Wally Moline
for serving on my thesis committee. I sincerely thanks Dr. Mike Allen, not only for
serving on my thesis committee, but for his valuable help.

Thanks are also due to Carol Daniel, Karen Chou, Jim Liesman, for their
assistance and to my fellow graduate students and friends. A special thanks to Luis
Solorzano for his friendship, support, encouragement and sharing of knowledge.

I am indebted to my parents, Sol and Leonel, and family for their many

sacrifices, encouragement, and continued love and support.

iv

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

Introduction

Literature review

Digestibility of tropical forages

Sulfur nutrition in ruminants

Sulfur metabolism by rumen microorganisms
Sulfur requirements

Nitrogen to sulfur ratio

Ammonia requirements

Isoacids for microbial protein synthesis _
Some aspects about VFA production in the rumen
Isoacid requirements

Materials and methods

Materials

Treatments and experimental design

In vitro rumen digestibility

Forage fiber analysis

Analysis of ammonia concentration

Hydrogen sulfide analysis

Determination of volatile fatty acid concentration
Statistical analysis

Results

Page
viii

ix

OOQUJUJHX

10
13
15
20
21
23
29
29
29
32
33
34
34
35
35
37

aaéfi<

Discussion
Summary
Conclusion
Appendix

List of References

67
71
72
73
86

Table

10

11

12

13

14

LIST OF TABLES

Sulfur requirements of ruminants

Calculated levels of NH3, H28 and isoacids in the fermentation
flasks

Composition of the solutions used in the basal media
Composition of the basal media

Volatile fatty acid concentration and true digestibility of tropical
forages after a 48 hour in vitro rumen fermentation

Analysis of variance of the effects of ammonia, sulfur and isoacids
on fiber fraction digestibility after a 48 h in vitro rumen
fermentation of eight tropical forages

Analysis of variance of the effects of ammonia, sulfur and isoacids
on volatile fatty acid concentration after a 48 h in vitro rumen
fermentation of eight tropical forages

Isoacids, ammonia and sulfur concentration after a 48 h in vitro
rumen fermentation of tropical forages

Effects of ammonia, sulfur and isoacids on the in vitro rumen
fermentation of Brachiaria decumbens

Effects of ammonia, sulfur and isoacids on the in vitro rumen
fermentation of Cenchrus ciliaris

Effects of ammonia, sulfur and isoacids on the in vitro rumen
fermentation of Cynodom dactylon

Effects of ammonia, sulfur and isoacids on the in vitro rumen
fermentation of Digitaria decumbens

Effects of ammonia, sulfur and isoacids on the in vitro rumen
fermentation of Gliricidia sepium

Effects of ammonia, sulfur and isoacids on the in vitro rumen
fermentation of Leucaena Ieucocephala

vii

12
30

31
32
38

39

48

53

57

58

59

62

63

15

16

Effects of ammonia, sulfur and isoacids on the in vitro rumen
fermentation of Panicum maximum

Effects of ammonia, sulfur and isoacids on the in vitro rumen
fermentation of Pennisetum purpureum

65

Figure

10

ll

12

LIST OF FIGURES

Page
The fate of sulfur in ruminants 7
Pathway of sulfur amino acid biosynthesis from sulfate and other 8
inorganic sulfur compounds
Ruminal degradation of proteins 17

The effect of the interaction of forage species and nitrogen on the 41
in vitro rumen true digestibility at 48 h of fermentation.

The effect of the interaction of forage species and sulfur on the in 42
vitro rumen true digestibility at 48 h of fermentation.

The effect of the interaction of isoacids and nitrogen on the in 43
vitro rumen true digestibility at 48 h of fermentation

Total volatile fatty acid concentration after a 48 h in vitro rumen 45
fermentation of eight tropical forages (pooled treatments)

Acetate concentration after a 48 h in vitro rumen fermentation of 46
eight tropical forages (pooled treatments)

Propionate concentration after a 48 h in vitro rumen fermentation 47
of eight tropical forages (pooled treatments) ‘

Effects of isoacids on total volatile fatty acid concentration after a 50
48 h in vitro rumen fermentation of tropical forages (pooled
treatments)

Effects of nitrogen level on acetate concentration after a 48 h in 51
vitro rumen fermentation of tropical forages (pooled treatments)

The effects of the interaction of isoacids and nitrogen on 54
propionate concentration after a 48 h in vitro rumen fermentation
of eight tropical forages

13 Effect of sulfur on the concentration of total isoacids after a 48 h 55n
in vitro rumen fermentation of eight tropical forages

VF A
BCVFA
C2

C3

C4

1C4

1C5

LIST OF ABBREVIATIONS

Nitrogen

Sulfur

Isoacids

Volatile fatty acid
Branchedcchain fatty acid
Acetate

Prepionate

Butyrate

Isobutyrate

Isovalerate

Valerate

Neutral detergent fiber
Acid detergent fiber
Lignin

Acid detergent fiber digestibility
Cell wall digestibility
Lignin digestibility
Cellulose digestibility
Hemicellulose digestibility
In vitro true digestibility

INTRODUCTION

Forages are an important source of feed for ruminants and other animals. In
tropical regions, pastures become the main if not the only source of feed available for
ruminants, horses and pigs. Tropical grasslands have the capacity to sustain animal
production based upon extensive farming systems. Even though tropical forages may
yield up to 22,000 kg - ha‘l - yr’1 of dry matter (DM) (Tinnimit, 1974), animal production
is seriously limited by the seasonal nature of pastures. In most tropical countries there
are two seasons: wet and dry. During the wet season acceptable quality and quantity
of forage is available. However, during the dry season not only the nutritional value
decreases but also the amount of pasture to graze is drastically reduced.

Tropical forages do not supply sufficient energy for the production of meat and
milk by ruminants. For dairy cows grazing tropical pastures, energy rather than protein
was found the first limiting factor for milk production (Delgado and Randel, 1989).
Most of the useful energy of tropical forages is obtained from carbohydrates. Their
energetic value depends, to a great extent, on the digestibility of the carbohydrate
fractions of cellulose and hemicellulose which are digested in the rumen by the action of
microorganisms (Van Soest, 1982). For optimal digestion of fiber, rumen
microorganisms need ammonia (NH), sulfur (S) and isoacids as well as other factors

such as vitamins and minerals.

2

There are several grasses and legumes of major economic importance in tropical
zones. A study of factors affecting ruminal fermentation of these forages is nwded in

order to find ways to enhance their economic value for ruminants.

REVIEW OF LITERATURE

D' i ’ ' f r i l f

The great potential of the tropics for animal production lies in the enormous yield
of biomass that can be produced per unit of land area. However, productivity per animal
in the tropics has remained low because pronounced wet seasons are almost always
followed by long dry periods. Dry pastures are generally low in both protein and
digestible energy (Preston and Leng, 1975). Of forages widely used in tropical regions,
signal grass (Brachiaria decumbens), buffel grass (Cenchrus ciliaris), star grass (Cynodon
dactylon), pangola grass (Digitaria decumbens), guinea grass (Panicum maximum),
elephant grass (Pennisemm purpureum) , leadtree (leucaena Ieucocephala), and shad
(Gliricidia sepium) are produced in the Dominican Republic.

It is common knowledge that digestibility decreases with increased maturity of
forage (Van Soest, 1982). Low digestibility is associated with lower leaf to stem ratios
and higher fiber contents (Panditharatne et a1, 1987). The apparent DM, CP, NDF, and .
ADF digestibility of guinea silage fed to wethers was greater for 2 than for 3 weeks of
plant growth. Chopping the grass before ensiling increased digestibility compared with
unchopped forage.

Dry matter digestibility and intake of tropical grasses are considerably lower than

those of their temperate climate counterparts (Minson and Bray, 1980). This is due to

4
the adverse effect of high temperatures in the tropics (McLeod and Minson, 1970).

Furthermore, tropical grasses have a higher cell wall content and lower dry matter
digestibility (DMD) than temperate grasses. Wilson and Hattersley ( 1989), worked with
leaves of 12 Panicum with C, (temperate) and C4 (tropical) photosynthetic pathways.
They reported lower cell wall content (37-49%) and higher DMD (67-74%) for C3 and
higher cell wall content (49-67%) and lower DMD (53-67%) for C4. Similar results
were obtained by Hill et a1. (1989) with C, and C4 Panicum species.

The occurrence of high photosynthetic capacity in the leaves of C4 plants confers some
advantages which enable them to adapt to certain ecological conditions better than C3
species (Spedding, 1971).

Fiber represents a significant fraction of the diet of herbivores. Consequently,
the animals productivity is limited by their ability to consume and digest the fibrous
portion of the diet. Allen and Mertens (1987) developed mathematical models to define
the processes of fiber digestion and for evaluating factors affecting digestion of fiber in
anaerobic systems. They found that the largest independent constraint on fiber digestion
is the fraction of fiber that is indigestible, which represents up to one-third and one-half
of the total fiber fraction of grasses and legumes, respectively. Van Soest (1973) stated
that the extent to which cell walls are digested depends on the lignin fraction which
determines the availability of cellulose and hemicellulose.

Tsai et a1. (1967) in their study of the effect of dietary fiber on lactating cows in
the tropics, observed an increased heat stress (measured as rectal temperature) as a result

of an increase in fiber intake. They concluded that fiber level should be considered in

formulating rations for dairy cows.

McLeod and Smith (1989) studied the effect of fiber level of forages on eating
and rumination behavior. It was concluded that when a ruminant is fed diets of high
fiber content, voluntary intake is not always reduced because of restrictions in either
rumen fill or rumination. Van Soest and Marcus (1964) examined 96 forages and found
that there was no significant relationship between cell wall constituents and voluntary
intake when forage cell wall content was less than 60% of the dry matter (including most
legumes and a few immature grasses). However, when values were above 60% there
was a marked decreased in voluntary intake with increasing content of cell walls.

Moir (1974) developed an equation to estimate the metabolizable energy from cell
walls and digested cell walls which could be used not only with a wide range of grasses,
but also appeared to apply to legumes. Dry matter digestibility (DMD), dry matter
intake (DMI) and fiber fractions differed between forage classes and animal species (Reid
et al., 1988). Also, C4 grasses were consumed at levels higher than would be expected
from their DMD and fiber concentrations. Ruminants appear to increase neutral
detergent fiber intake (NDFI) in response to higher NDF concentrations in the forage.
McLeod and Minson (1988) found no difference in breakdown between temperate and
tropical forage when using a digestion-detrition simulator for 48 h digestion. The
apparatus simulates digestion and detrition (rubbing) in the rumen. They reported that
both digestion and detrition reduced forage particle size in vitro.

A study of the chemical composition and digestibility of 101 tropical grasses was

conducted by Kayongo-Male et a1. (1976). Grasses were harvested at 30 days of growth.

6
Wide ranges in the percentage of NDF (45.7 to 79.2%), ADF (30.9 to 45.3%), and

hemicellulose (11.7 to 37.5 %) were found. Digestibility estimates obtained by the Tilley
Terry method and NDF digestibilities ranged from 42.6 to 66.0% and 22.0 to 62.0%,
respectively. ADF seemed more important than lignin in determining digestibility values.
It was pointed out that the significance of Lignin/ADF ratio in relation to digestibility
estimates was much less for in vitro estimates than for estimates calculated from
predictive equations. They concluded this indicated that lignification of cellulose had less
influence on digestibility of tropical than of temperate forages.

Butterworth (1964) reported that digestible energy of twenty-four forages ranged
from 2.39 kcal/g for pangola silage to 3.08 kcal/g for signal silage. In addition, no
correlation was demonstrated between the content of crude fiber or crude protein and the
digestible energy of the forages.

Ishizaki et a1. (1976) found a positive correlation between the in vivo (65.4) and
in vitro (68.2) digestibility of pangola grass by sheep.

Dry matter digestibility was 59.7 and 66.7% for leucaena and buffel, respectively,
when lambs were fed a diet containing sisal pulp, sisal bagasse and urea (Yerena et al.,
1978). Child et a1. (1982) conducted a digestibility study with heifers using the nylon
bag technique and found that the mean rumen digestion index (dry matter disappearance
using the nylon bag technique) for ground leucaena samples varied from 13.6% for
shattered pods to 90.6% for the small developing pods. The index for crude protein
varied from 5.2 to 31.7. It was concluded that leucaena can serve as a high quality feed

for livestock.

Gut

 

 

 

 

 

 

Intake -——§ Reticulo-rumen {—4 I Post ruminal tract F——>Faeces
I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SS
1 4r
Blood and extra-cellular fluid W001
Hair
Inorganic and organic sulphur Milk
I“ so.“
S-
I Liver I Kidney Urine
Inorganic Organic
cg. Sulphates cg. Amino acids
Proteins, Esters

 

 

 

Tissues

Figure l. The fate of sulphur in ruminants. Adapted from Bray and Till (1975).
ri i in min n

Interest in S metabolism began when du Vigneaud demonstrated, by using
radio—labeled cystathionine, that mammals convert methionine to cystine (Garrigus,
1970). The S-containing amino acids are important components of many proteins,
enzymes, vitamins and several hormones. Sulfur plays a major role in protein structure
(Johnson et a1. , 1970). It is a component of two essential amino acids, cysteine and
methionine. Therefore, S is required by the ruminant to synthesize S-containing amino

acids within the rumen. The fate of S in ruminants is illustrated in Figure 1.

8

The presence of rumen microorganisms permits the ruminant to utilize fibrous
material and forage plants as sources of dietary nutrients. The microbes ferment forage
to volatile fatty acids, and convert inorganic N and S to microbial protein.

Orskov (1982) found that microbial biomass may contain as much as 8 g of S/kg
DM, found mainly in the protein fraction. Sulfur in feed is reduced to sulfide (H28) in
the rumen and incorporated into microbial protein or absorbed directly as HZS. Sulfide
is the key intermediate between the breakdown of ingested and recycled S and its
utilization or loss from the ruminant system (Bray and Till, 1975).

The general pathway of S amino acid biosynthesis from sulfate and other

inorganic S compounds is presented in Figure 2 (Roy and Trudinger, 1970).

 

cysteine
/’ it
SO. " * é} SO,‘ " {=} S‘ {=} cystathione {=} methionine
\ / It //
8,0, \ homocysteine

 

Figure 2. Pathway of sulfur amino acid biosynthesis from sulfate and other inorganic sulfur compounds.

I' 11 mi
Rumen bacteria rapidly reduce inorganic S and incorporate it into organic
compounds. The optimum pH for the reduction of sulphate is 6.5 (Kandylis, 1984).

However, the capacity of the reticulo-rumen system for sulphate reduction is partially

9
dependent on a period of adaptation to dietary S (Bray and Till, 1975).

The microbes can utilized both inorganic and organic S to synthesize S-containing
compounds available for absorption (Kandylis, 1984). If dietary S is inadequate,
microbial activity is slowed. Sulfur losses may occur because of the formation of volatile
H28 (Goodrich and Garret, 1986).

Emery et a1. ( 1957a and b) conducted an in vitro study with labelled sulfate and
substrates representative of concentrate and forage rations. They observed that cysteine
formation was twice as rapid as methionine formation and sulfate incorporation into
amino acid was more rapid with forage as the substrate.

Pittman and Bryant (1964) observed that-some strains of Bacteroides ruminicola
required cysteine and methionine. Sulfate reduction in the rumen is executed by both
assimilatory and dissimilatory microorganisms. Assimilatory reduction involves sulfate
reduction to sulfide with accompanying incorporation into cellular materials.
Dissimilatory microorganisms reduce sulfate without using it, producing free hydrogen
sulfide (Peck, 1970).

Moir (1970) reported that in vitro incubations fermenting cellulose with 358,
methionine and cysteine accounted for 28 and 34 % of the S-protein produced, while .
sulfide accounted for 88% . Hume and Bird (1970) also reported accumulation of sulfide
in the rumen following sulfate administration.

Sulfide is probably the central metabolite in ruminal S metabolism. Therefore,
dietary S utilization by rumen microorganisms depends on the quantity and source of

available S, as well as on the loss of sulfide from the rumen (Moir, 1970). The relative

10

rate of sulfide formation from S compounds is as follows: cysteine > inorganic
S > methionine (Bray and Till, 1975). Bray and Hemsley (1969) reported ruminal
sulfide levels of 0, 2, and 6 pg sulfide! ml of rumen fluid in sheep fed diets containing
.06, .14, and .32% S, respectively. However, Hume and Bird (1970) found values for
rumen sulfide of .5, 1.9, 4.3, and 3.5 pg sulfide/ml when consuming a diet containing

either 0.08, 0.2% sulfate S or 0.2% cysteine S and 0.4% sulfate+cysteine S,

respectively.

Wm

To obtain a well functioning rumen, rumen microorganisms need to be supplied
with adequate amounts of S. Different species may have different requirements
depending on a variety of factors such as age and condition of the animals, natural diet
vs. purified diets, source of S and dietary N, or in vitro vs. in vivo experiments.

Bird (1972a) showed that less dietary S is required by cattle than sheep, and
cattle may tolerate a wider N :8 ratio in the feed than sheep. Apparently, this is because
S is recycled more effectively in cattle. In another study, Bird (1972b) found that for
sheep a small increase in S intake (0.36 g/d) improved the nutritive value of a S
deficient, low protein roughage (oat hull) diet in which urea was supplied. Earlier work
performed by Thomas et al. (1951) showed that growing lambs fed semi-purified rations
with less than 0.1% S and supplemented with urea had improved rates of gain and N
retention after NaZSO4 was added to the diet.

Purified diets with and without S and containing urea as the source of N fed to

l 1
sheep resulted in decreased intake and weight loss in the S deficient animal (Whanger and

Matrone, 1970). Moreover, it was found that gram positive organisms were predominant
in the rumen of sheep fed S adequate diets while gram negative bacteria predominated
in the rumen of S deficient sheep (Whanger and Matrone, 1970). Hence, there was an
accumulation of lactate in the rumen of sheep fed the low 8 diet, and just traces in the
rumen of the S fed sheep. In addition, there was more butyric and higher fatty acids
when S was adequate while more acetate and propionate was found when S was deficient.

Several studies were conducted by Bouchard and Conrad (1973a, b and c)
concerning the requirements of S by lactating dairy cows. They found that dietary S of
.12 % and .18 % produced a zero and a positive S balance, respectively. Therefore, they
concluded that those levels should approximate the limits of S requirements in lactating
dairy cows. They also compared the availability of different sources of dietary S.
Sodium and calcium sulfate provided an availability of S of about 77 to 87% . However,
S from lignin sulfonate was poorly digested (42 to 53%). Dietary S requirements and

variation within species and physiological stage of the animal are presented in Table l.

12

Table 1. Sulfur requirements of ruminants.

 

 

Ruminant Sulfur, % DM Reference
Growing-finishing cattle 0.10 Goodrich & Garret, 1986
Growing-finishing sheep 0.14—0.26 Goodrich & Garret, 1986
Calf starter concentrate 0.20 NRC, 1989
Growing heifers 0.16 NRC, 1989
Dry pregnant cow 0.16 NRC, 1989
Lactating cow 0.20 NRC, 1989
Mature bulls 0.16 NRC, 1989

 

Generally, rations composed of natural plant and animal components usually
contain adequate S to meet requirements of ruminants. However, some grasses are low
in available S (Johnson et a1. , 1970). Animals fed low quality forages may respond to
S supplementation (Goodrich and Garret, 1986). Rees et a1. (1974), reported an
increased digestibility in sheep fed pangola grass supplemented with S. However, an
increased voluntary intake of 44 % was found when the same grass was fertilized with S.
Bray and Hemsley (1969) found that sulphate supplementation increased crude fiber
digestion and N and S retention when added to a poor quality forage diet fed to sheep.
In another study, Guardiola et al. (1983) observed increased total fiber digestibility in
lambs fed low or high quality forages supplemented with sulfate or methionine. Kennedy
and Siebert (1972) and Kennedy (1974) studied the effect of sulfate additions to rations
composed of tropical spear grass and urea in sheep. Sulfur supplementation resulted in

improved dry matter digestibility, N retention and feed intake. Inorganic S added to the

l3
rumen can be absorbed directly into the blood (Kennedy and Milligan, 1978). Serum

inorganic sulfate-S were increased to a maximum of 35—46 mg/L by infusion of sulfate

into the rumen or abomasum of sheep given bromegrass.

WW

There is a close association between S and N in both plant and animal cells.
Most diets that contain required levels of protein will also provide adequate levels of S
(National Research Council, 1989). Poor quality diets supplemented with Non-Protein
Nitrogen (NPN) will require additional S supplementation (Goodrich and Garret, 1986).
The relative proportion of S to N in these diets is important.

The N :8 ratio of body tissue is about 15:1 (Garrigus, 1970). According to Bray
and Till (1975), it could be argued that the ratio of N to S retention should be of the
same order as the ratio in body tissue. This is true for cattle. However, sheep have a
larger S requirement for wool production, requiring an overall N:S ratio of 13.5:1 (Bray
and Till, 1975). Hume and Bird (1970) found a microbial protein N:S ratio of 18: 1.
For ruminal bacteria, the ratio of total N :S has been reported to range from 11:1 (Moir,
1970) to 22:1 (Bird, 1972a). Nitrogen to S ratios of feedstuffs range from 10:1 for most
cereal grains, 18.2:1 for legumes (e.g. peanut meal) to 26.9:1 for zein (Moir, 1970).

A close relationship has been observed by several investigators between dietary
N and S content in ruminant diets. The ratio of the two elements is used as a guide to
recommend proper levels of supplemental S. Bray and Hemsley (1969), using a simple

oat hull, urea, and mineral diet with sheep, found dry matter digestion increased from

14
46.6 to 51.9% when sulfate-S intake was increased to narrow the N:S ratio from 24 to

9.7:1. Moir (1970) described the relationship between N and S intake and N balance.
They found that the maximum N balance was achieved with a dietary N :8 ratio of 10: l.

The National Research Council (1989) estimated the S requirement for lactating
cows at 0.20% of the total diet. This implies a N:S ratio of 12:1. Slyter and Weaber
(1971) reported that calves fed adequate S were efficient in retaining N. Saliva appears
to be the major source of recycled S (Kandylis, 1983). The N:S ratio in the residual-S
(protein) fraction of the saliva remains relatively constant at 11-12:1.

Adequate S supplementation can be achieved with methionine, elemental S or
sodium sulfate (Johnson et al. , 1970). However, supplemental S may be less available
than the S source found in the natural diet (National Academy of Sciences, 1976).
Organic sources are preferred such as D-L-methionine. Inorganic sources like sodium
sulfate and elemental S are the least available (Goodrich and Garret, 1986). However,
Onwuca and Akinsoyinu (1989), reported that elemental S supplementation to small
ruminants improved dry matter intake, live weight gain and N utilization.

The S for the microbes is generally derived from degradation of dietary protein,
and therefore a deficiency in S is likely to occur only if there is a deficiency of N. Such
a deficiency will lower the number of lactic acid fermenting microorganisms in the
rumen (Johnson et al. , 1970). Sulfur deficiency may result in reduced milk production
and weight gain, anorexia, low dry matter digestibility, profuse tearing and salivation,
dullness, emaciation and in extreme cases, death (Goodrich and Garret, 1986). Kandylis

(1984), reported that S toxicity may occur if dietary S exceeds .3 to .4% of the D.M.

15

l . . |

Despite the amino acid needs of the ruminant animal, there are N requirements
for the rumen microbiota especially when the animal is expected to use forage and other
cellulose containing energy sources (National Academy of Sciences, 1976). Because of
the limitations in quantity of protein synthesized by the rumen microbiota, it becomes
necessary to provide adequate dietary protein or provide all other intermediates essential
for microbial protein synthesis (Chalupa, 1973).

Studies on the nutrition of ruminal cellulolytic bacteria emphasize the importance
of their interaction with other microbial species to synthesize chemicals such as
B-vitamins, ammonia and certain VFAs often essential for bacterial growth. The major
microbial nutrients include minerals, S, and N, which leads to ammonia formation
(Bryant, 1973).

Ammonia is a vital ingredient in microbial synthesis (National Academy of
Sciences, 1976) and is produced by ruminal microbes from both protein and NPN
substances (Allison, 1969). It is not the only nitrogenous nutrient required for ruminal
microbial growth (McDonald, 1948), but is the main one (Allison, 1969; Hungate,
1966). Bryant and Robinson (1961) studied the N requirements of some cellulolytic I
bacteria and found that ammonia was utilized as the sole source of N by different strains
of Ruminococci sp.

Other sources of ammonia in the rumen include urea from blood, salivary
proteins, epithelial cells sloughed from the mouth, esophagus and ruminal epithelium

(Nolan et a1. , 1973). Supplementation of ammonia to meet optimal ammonia

16

concentrations in the rumen depends on the amount of ammonia which can be degraded
from dietary components, the amount of recycled endogenous urea and levels of other
components like energy and minerals. The availability of energy from different sources
is a key factor in evaluating effects of supplemental ammonia in the ruminant. Pidgen
(1971) as cited by National Academy of Sciences (1976) report that the lignocellulose
complex accounts for most of the energy in mature forages. Nitrogen composition of
roughages will affect their individual rate of digestion. Of the N in fresh forage 70-90%
is in protein (Waldo, 1968) in the sense that it can be made insoluble by denaturation.
The 10-30% of the N that is soluble is often considered NPN and contains nucleic acids,
peptides, amino acids, amines and purines and occasionally nitrate (Spedding and
Diekrnahns, 1972 as cited by Tamminga, 1986). In fresh grass, total N content is
usually high and a large proportion of it is rapidly degraded in the rumen (Tamminga,
1986). Diets based on fresh grass contain low amounts of energy and a surplus of rumen
degradable protein. In order to utilize NPN efficiently with such a diet, supplementation
with an adequate energy source to favor microbial growth is required (Tamminga, 1986).
Russel and Hespell (1981) indicated that insufficiency of peptides, amino acids, and
branched chain fatty acids at certain times after feeding may be a major factor causing
energetic uncoupling, resulting in continued production of fermentation products without
concomitant bacterial growth in the rumen. A scheme for ruminal degradation of

proteins is presented in Figure 3.

17

 

DIETARY AND OTHER PROTEINS

POLYPEPTIDES
AMINO ACIDS + SHORT PEPTIDES + NH, + co,
_, ACETATE, rsonurmra
B o o H
_) 2-METHYLBUTYRATB -> MICRO “L R WT
ISOBUTYRATE
—>
mg + co, _, AMINO Acrns

 

Figure 3. Ruminal degradation of proteins. Adapted from Russel and Hespell (1981).

Different sources and levels of ammonia precursors need to be considered. Urea
is the main NPN source of N used for microbial protein synthesis. Other NPN products
such as urea-carbohydrate, ammonium salts, ammoniated molasses, biuret etc. have been
used as N sources for ruminants.

According to the National Research Council (1989), N is involved in the rumen
in two ways. There is an efflux of N from the rumen which occurs through absorption
and passage of ammonia, and an influx of N to the rumen through the diffusion of blood
urea (Houpt and Houpt, 1968) and the secretion of salivary urea (National Research

Council, 1989). Dietary NPN must be first transformed to ammonia in order to be

18
utilized for microbial growth. NPN is used most efficiently for rumen protein

production when it produces an ammonia concentration that is optimal for bacterial
protein synthesis (Chalupa, 1973). Some species of ruminal bacteria use exogenous
amino acids (Allison, 1969). However, amino acids in peptides are more efficiently
utilized than are free amino acids by other species (Pittman and Bryant, 1964). More
recently, Cotta and Russel (1982) observed that high concentrations of peptides and
amino acids resulted in high yields of bacterial protein but conversion of free amino acids
to microbial cell protein was poor.

Hume et al. (1970) reported that the addition of NPN supplements to ruminant
rations increased microbial protein synthesis while ammonia concentration remained low
and constant up to a dietary N intake of 9 g/d. However, diets with a higher N intake
(16 g/ d) produced no further increase in rumen protein output and ammonia concentration
increased. Satter and Slyter (1974) showed that ammonia in excess-of 5 mg NH3-N/dl
of ruminal ingesta had no effect on fermentation rate. Rumen bacteria can scavenge
ammonia from low concentration environments, but when ammonia starts to accumulate
bacterial growth is not enhanced by providing 7 additional NPN (Chalupa, 1973).
Therefore, once ammonia concentration reaches 2 to 3 mg NH3-N/dl, microbial needs
are satisfied (Roffler and Satter, 1975a). However, because of its variation in the rumen,
maintenance of an average concentration of 5 mg NH3-N/dl is recommended (Roffler and
Satter, 1975b). The same authors in a lactation study, observed that NPN
supplementation did not improve milk production if the ration contained more than

12.5% CP or more than 4 mg NHg-N/dl rumen fluid.

’ 19

Huber and Kung ( 1981), in a review of the protein and NPN utilization in dairy
cattle, explained that with increasing dietary N, rumen ammonia increases more rapidly
with NPN than natural protein. Schaefer et a1. (1980) determined that ammonia
saturation constants for the predominant species of ruminal bacteria were less than 50 11M
and that organisms growing in a medium of 1 mM (1.7 mg/dl) ammonia should achieve
95 % of their maximum specific growth rate, but would not necessarily provide for
maximum yields of bacterial cells. However, Bull et a1. (1975) as cited by Huber and
Kung (1981) showed an increased in microbial protein production until rumen ammonia
reached about 20 mg/dl. In vivo studies suggested that synthesis of microbial protein is
not maximized until rumen ammonia reached 10 (Hume et al., 1970) to 29 (Miller, 1973)
mg/dl.

Mehrez and Orskov (1977) showed that NH, concentrations in the rumen needed
to be much higher frorn what had been reported. At 95, 85, and 75% of maximal rate
of substrate disappearance, NH, in the rumen digesta was 24, 19, and 15 mg/dl. When
ruminal NH, was 7 mg/dl, only 50% of the maximal rate of breakdown was achieved.
Ruminal NH, concentrations rise after consumption of a meal and net absorption is
positively correlated with ruminal concentrations in goats, sheep, and cattle. Because of
gut fermentation, a substantial portion (16-80%) of N is absorbed as ammonia N
(NH,-N). Net uptake of NH, is higher with forage diets than with high energy diets
(Huntington, 1986).

Ammonia toxicity could be produced when urea is consumed in large quantities

in a short time. Signs of toxicity include uneasiness, dullness, muscle and skin tremors,

 

 

20

excessive salivation, frequent urination and defecation, rapid respiration, incoordination,
tetany and death (Bartley et al. , 1976). Apparently, high rumen ammonia concentrations
may exist without producing toxicity if the ration is readily ferrnentable and rumen pH

is below 7.4 (Bartley et al., 1976).

WWW

It is well known that the microbial biomass consists of a multitude of microbes
of different species (Hungate, 1966). Ruminal bacteria are by far the most frequently
occurring group of organisms, although they do not always constitute the greatest
biomass of the rumen microorganisms (Harrison and McAllan, 1980). Among them, the
cellulolytic bacteria give the ruminant the ability to survive on poor quality fibrous
forages (Orskov, 1982). Cellulolytic bacteria are strictly anaerobic and besides requiring
ammonia and S, their growth rate is dependent on the presence of branched chain volatile
fatty acids (Dehority et al., 1967; Bryant and Doestch, 1955; Allison, 1969) such as
isobutyrate and isovalerate (Allison and Bryant, 195 8). These branched chain acids
appear to be formed mainly by degradation and deamination of branched chain amino
acids from dietary protein by some of the non cellulolytic bacteria and ciliate protozoa
(Slyter and Weaver, 1971). The branched chain volatile fatty acids cannot be synthesized
by most rumen cellulolytic bacteria (Allison et a1. , 1974). Therefore, protein synthesis
may be limited by the supply of these nutrients in diets containing low dietary protein
(Hume et al., 1970). Slyter and Weaver (1971) in an in vitro study with several strains

of cellulolytic bacteria, found branched chain fatty acids were formed by a mixed rumen

21
population fed a diet with no amino acids and rapidly fermented carbohydrates.

However, branched chain fatty acids have been reported to increase total nricrobial
synthesis (Hume, 1970a and b) and N retention in ruminants (Oltjen et al., 1971),
although a beneficial effect has not always been attained (Cline et a1. , 1966).

Naga and Harmeyer (1975) studied the relationship between the production of VFA and
synthesis of microbial protein in vitro. They observed a negative correlation between

microbial growth and end products formed.

WWWm

Large quantities of volatile fatty acids, particularly acetic, propionic and butyric
acids, are produced in the rumen by microbial fermentation of dietary carbohydrates and
protein and are absorbed into the bloodstream mainly through the rumen wall (Barcroft
et al., 1944), constituting the major portion of absorbed energy (Bush et al., 1979).
Early research (Masson and Phillipson, 1951; Kiddle et al., 1951) was conducted to
study the concentration of these acids in blood leaving the rumen compare to its
concentration in the rumen itself. Production of VFA is affected by the type and amount
of plant material as well as by pH in the rumen (Van Soest, 1982). I

Weller et a1. (1967) measured by continuous infusion of 1‘C labelled VFA, the
total and individual VFA production in sheep fed luceme hay. This study showed that
the composition of the acids initially found in the rumen were 77-83% acetic , 15-18%
propionic, and 1-7% butyric . Similar results were obtained with sheep fed different

diets (Leng and Brett, 1966) and with grazing sheep (Leng et al., 1968). This last study

22

demonstrated a method to make comparisons between pastures on the basis of their
potential yield of energy for sheep, by determining VFA production rates in grazing
animals. They developed an equation to predict the amount of energy supplied by the
acids.

In their studies of VFA metabolism in sheep, Krishna and Ekern (1974a and b)
reported that the total amount of VFA produced when timothy hay was fed ranged from
3.80 to 3.93 monay or about 40—41% of the total metabolizable energy consumed. This
agreed with previous findings reported by Marston (1948) as cited by Knox et a1. (1967)
that volatile short-chain organic acids may provide ruminants with 40-70% of their
energy needs. Results of a short term study involving VFA intraruminal infusions
indicated a higher efficiency of utilization for propionic acid than for acetic (Armstrong
et a1. , 195 8). Weller et a1. (1969) measured the concentration of VFA by isotope
dilution when sheep were grazing pastures. The VFA production in the rumen was found
to increase during the period of growth of the pasture and to decline when it dried off.
Gray et a1. (1965, 1967), also conducted a series of experiments to study the production
of VFA in the rumen of sheep. They observed the same rate of VFA production with
different DMI of the same fodder ration. They found that the energy of the VFA
produced in the rumen was equivalent to about 54% of the digestible energy of the diet.

The interconversion of acetic acid into butyric acid by sheep was reported to be
between 50-80% when lucerne hay was fed (Weller et a1. , 1967) and from 51-66% if fed

dried grass cubes (Bergman et al., 1965).

23

W

Rumen bacteria are unique in their ability to synthesize amino acids by first
carboxylating short chain acids to form alpha-keto acid analogues of amino acids and
then, to utilize ammonia to form the complete corresponding amino acid. Isovalerate,
isobutyrate and 2-methylbutyric acid are used for the biosynthesis of leucine, isoleucine
and valine, respectively, as well as for synthesis of higher branched—chain fatty acids and
aldehydes (Allison et al., 1962; Allison and Peel, 1971; Allison et a1, 1974; Robinson
and Allison, 1969). These branched-chain fatty acids and the straight-chain valeric acid
stimulate growth and activity of cellulolytic and some noncellulolytic bacteria (Allison
et al., 1962; Bryant, 1973; Dehority et al., 1967).

The addition of isoacids to ruminant rations have shown a positive effect on
performance (Cline et al., 1966; Felix et al., 1980; Hemsley and Moir, 1963; Papas et
al., 1984), dry matter digestibility (Soofi et al., 1982), microbial growth (Cline et al.,
1958; Gorosito et a1. , 1985), insulin production (I-Iorino etal., 1968), microbial protein
synthesis (Hume, 1970a and b) and N retention (U munna et al., 1975; Felix et al.,
1976).

Low availability of isoacids limits ruminal fermentation, especially with high
roughage diets, or high feed intake and high energy demand which is the case in lactation
(Cook and Towns, 1987). Gorosito et a1. (1985), observed an increase in cell wall
digestion by ruminal bacteria supplemented with isoacids. Quispe (1982), conducted
experiments where rumen acetate production was measured to determine the rate of

fermentation. Her work showed that isoacids increased acetate production in sheep fed

24

pineapple tops. Later, Kone (1987) investigated the interaction of isoacids and Monensin
on ruminal fermentation, observing that isoacids at 15 mg/dl increased acetate and VFA
production while Monensin reduced acetate and VFA, but increased propionate
production. The combination of both, increased acetate but did not eliminate the effect
of the ionophore on propionate.

Cline et a1. (1958), attempted to determine the nutrients needed for rumen
microbial growth in vitro. By changing the carbohydrate source (starch, glucose and
cellulose) and levels of urea, they found that as urea increased, the level of valeric acid
decreased and cellulose digestion increased. This suggested that as microbial growth
increased, the rate of utilization of valeric acid increased.

Previous work has shown that the addition of a mixture of four- and five-carbon
branched chain and straight chain VFA to low protein diets may lead to an increase in
the utilization of such a diet by ruminants. The addition ‘of a mixture of branched chain
VFA to sheep fed a protein free purified diet adequately supplied with NPN, increased
protein production from 71 to 81 g/ day. In another study , Hemsley and Moir (1963)
found that the intake of a milled oaten hay diet was increased by the addition of 0.56%
of a mixture of isobutyric, isovaleric, and n-valeric acids. Later, Cline et a1. (1966),
reported a significant increase in N retention by lambs supplemented with 4.18 g
isobutyrate, 1.18 g n-valerate, and 5.9 g isovalerate per day. Studies conducted by
Umunna et a1. (1975), showed that feeding or rumen infusion of isobutyrate and/or
isovalerate to lambs on high roughage, urea supplemented rations improved N retention

and decreased urinary N loss. Rumen ammonia and blood urea were not affected by the

25

acids.

Low concentrations of isovalerate, 2—methylbutyrate and peptides in the diet can
improve efficiency of synthesis of rumen bacterial protein from soluble carbohydrates up
to 11.2% and 16.4%, respectively (Russell and Sniffen, 1984). Cummins and Papas
(1985), reported that the addition of isoacids (C, and C,) to a corn silage based diet,
increased dry matter digestion and microbial growth in vitro regardless of the dietary
crude protein content (13 to 16%). Soofi et al. (1982), measured the in vitro effects of
branched—chain VFA , urea, starch and trypticase, and their interactions on dry matter
disappearance of soybean stover finding a positive effect of branched-chain VFA on DM
disappearance. The interaction of branched-chain VFA and tryptiease appeared to
provided a balance medium for bacterial growth.

Hefner et a1. (1985) studied the effect of branched-chain VFA supplementation
to corn crop residue diets. Dry matter and fiber digestibility tended to be higher when
lambs were fed natural protein supplements, while urea and branched-chain VFA tended
to decrease the extent of digestion. Gorosito et a1. (1985), incubated mixed ruminal
bacteria in an artificial medium with isolated plant cell walls and intact forages. They
added an equimolar mix (.30 mM) of C, and C, acids and observed that cell wall
digestion was increased 26.4 % . The individual isoacids were equally as effective as the
mixture, increasing cell wall digestion 25.4 to 26.6% . Valerie acid alone did not affect
cell wall digestion.

Earlier work conducted by Falen et a1. (1968) attempted to determine the effects

of soybean meal (SBM), urea and phenylacetic, 2-methylbutyric, isobutyric and isovaleric

26

acids on the intake and digestion of low quality roughages. All treatments substantially
increased intake, with isobutyrate giving the best response and isovalerate giving the
lowest increase in VFA production. More recently, Varga et al. (1988) conducted an in
vitro fermentation of diets containing SBM treated with 0.3 % formaldehyde, or
unheated. Formaldehyde treated SBM depressed fiber and protein digestion as well as
VFA and microbial production. However, N provided as urea and carbon skeletons as
isoacids restored fiber digestion.

An in vivo study conducted by Lassiter et a1. (1958) indicated that the
combination of isovaleric and valeric acids exerted a beneficial effect upon the growth
rate of dairy heifers fed a low quality roughage. Research by Felix et al. (1976)
suggested that mixtures of C, and C, branched-chain acids plus valeric acid improved N
retention, milk production, and persisteney of milk yield in dairy cows fed diets
containing urea, corn silage, and corn grain. Isoacid fed cows could achieve an increase
in milk production of 10% without any increase in feed intake (Papas et al., 1984).

The addition of salts of VFA to ruminant diets have been extensively studied.
Rogers and Davis (1982) found that intraruminal infusions of mineral salts of VFA to
steers reduced the molar percentage of ruminal propionate and increased that of acetate
when high grain diet was fed. However, no effect was obtained when salts of VFA were
added to a high roughage diet. Supplementation of diets for Holstein cows with
ammonium salts of branched-chain VFA increased milk production by 8 to 10%
(Peirce-Sandner et al., 1985) as cited by Otterby et al. (1990). Rogers et al. (1989),

reported the milk production response of cows fed calcium or ammonium salts of

27
branched-chain VFA early in lactation was higher (7.9%) than the response of cows in

mid- or late lactation. Dose response studies of dairy cows to 0, .4, .18, 1.2, or 1.6%
ammonium salts of branched-chain VFA added to the concentrate portion of the diet with
a forage:concentrate ratio of 50:50, 60:40, and 70:30 for first, middle, and late
lactation, respectively. It was found that supplementation of ammonium salts of
branched-chain VFA increased milk and milk protein yield during mid and late lactation.
A tendency to gain less body weight was also observed (Otterby et al., 1990).

The addition of sodium and calcium salts of branched-chain VFA to high grain
diets of small ruminants resulted in higher weight gain than to high roughage diets
(Orskov and Allen, 1970).

With respect to the utilization of sodium and calcium salts of VFA by small
ruminants, Orskov and Allen (1970) observed that addition of these salts to a high
roughage diet promoted lower weight gains than to a high concentrate diet. Later
research conducted by Poole and Allen (1970), showed that a mixture of sodium and
calcium salts of acetic acid added to low (40%) and high (85 %) concentrate diets
exhibited a greater live-weight, empty body-weight and carcass-weight gain than lambs
given unsupplemented diets. Response of weight gain to increasing levels of acetate salts .
was linear.

Among the factors affecting ruminal volatile fatty acid production, it was found
by Peters et al. ( 1989) in an in vitro mixed bacterial population that total production of
microbial products was greater at high than at low pH. Elliot et a1. (1987) recently

studied the influence of anaerobic fungi on rumen VFA concentration in viva, indieating

28

an increased concentration of rumen propionic acid as a result of the removal of the
rumen anaerobic fungi (RAF). This suggests that RAF may play an important role in
the fermentation of high fiber diets.

As stated by Bergen (1979), the ruminal fermentation is a coupled process
between carbohydrate degradation and microbial cell synthesis. Therefore, several
factors such as ammonia, branched-chain VFA, S and carbon chains are required to be
available at the same time in order to allow the optimal fermentation (Bergen and
Yokoyama, 1977). The advantages of supplementing isoacids, S and a NPN source to
ruminants have already been discussed. However, little information has been reported
concerning the interaction among those three factors. Recent reports (Quispe et a1. ,
1991; Brondani et al., 1991) indicated that in high fiber, low protein rations, S and N
requirements have to be met before isoacids can elicit increases in microbial growth and
cellulose digestion.

In the present study, an in vitro ruminal fermentation trial was carried out in
order to evaluate the effect of the interactions of S, N and isoacids on high fibrous

forages which are of common usage in Latin American countries.

MATERIALS AND METHODS

Materials

An in vitro experiment was conducted to investigate the effect of N11,, isoacids
and S on rate of fermentation of eight tropical forages. The forage samples collected in
the Dominican Republic were Brachiaria decumbens, Cenchrus eiliaris, Cynadan
dactylan, Digitaria decumbens, Gliricidia sepium, Leucaena leucacephala, Panicum
maximum and Pennisetum purpureum. The stage of maturity of the forages was 35 days,
with the exception of Leucaena (80 days), and Panicum (112 days). Samples were dried

overnight at 60°C in an oven and ground in a Wiley mill to pass through a 1 mm screen.

11 ' n i
Eight forages species in a 23 factorial design were studied. The factors were at
two levels each of N11,, S and isoacids. (Table 2).
The combinations of the three factors at two different levels resulted in 8
treatments (LLL, LLH, LHL, LHH, HLL, HHL, HLH and HHH) where NH,, 8 and
isoacids are the first, second and third factor, respectively, and L and H represent low

and high levels . All treatments were tested using each one of the 8 forages.

29

30
Table 2. Calculated levels of NH,, HZS and isoacids in the fermentation flasks.

 

 

Levels (NH,)1 (H28)l Isoacids
Low 5 mg/dl 2 mg/dl 0
High 10 mg/dl 6 mg/dl 15 mg/dl

 

‘ The levels of NH, and S achieved in the incubation mixture was 4.04 and 8.08 mg
NH,/dl for low and high NH,, and 1.36 and 4.1 mg H,S/dl for low and high S,

respectively.

The levels chosen for the three factors were based on reports from previous
experiments (Kane et al., 1989; Felix et al., 1980; Quispe et al., 1991). An equimolar
mixture of isoacids 6 g/dl (isobutyric, 2-m-butyric, isovaleric and valeric) was prepared
and neutralized with KOH, to pH 7.00. To obtain the final concentration required in the
fermentation (15 mg/dl) a dilution of 400 fold was made.

Different levels of S were provided in the reducing solution by varying the
amount of S (Na,S.9H,O). Ammonia was provided in the buffer solution as ammonium

carbonate (NILHCO,). The levels are shown in Table 3.

31

.88: 382.; a... «5.80 s 8.2.8... .

 

 

 

 

 

 

 

 

 

 

 

 

 

2a a: 2... mm. n|«. a
« « « « « .... 22.2 2 .
2a a... 0.2.6.358:
2. 2. 2. 2. 2. .6 o...

225%...

.. a a a a g
. . . . . « 0.5.68
2 2 2 2 2 u 0.21.2.2
«.2 «.2 «.2 «.2 «.2 « 6.5.65

22.5.5... 4%

a... g
««.c a... ««.o ««.c a .6»:
«a «.e «a «a «a a 6.3.2
«a «.m «a «.n «a m 62.2
82 82 .8. .8. .8. .6 ca:

225...th 3.2.2232

88 8.. 8.. one as hiolofizl
82. 82 m8 8... 2... a... .8222
.8. .8. .8. 82 :8. .a 0.:

225.42... 52....
.2»... 2. a... .3»... a a... .22.. a a: .2»... « a... .2»... « 3. 92.5325
.2»... 8 .22 .2»... 2 .22 .2»... a .22 .3»... 2 .22 .2»... n .22

 

538...... one... an: a .3... «.3... a...

882.... 5.3.8 a... 2.... .2: .5 r... J... .2... a... .88.... .a 38......an 32...... o... .e «.3... .8... o... a. .3... 38...... a... .a 82.858 .n 2......

32
E .2 I. ”.1“

Rumen fluid was collected from a Holstein cow that had been fed a wheat straw
diet for several weeks, blended for 30 seconds in a steel blender at low speed, passed
through cheesecloth and then glass wool under C0,. Fermentations were conducted in
125 ml Erlenmeyer flasks containing 0.5 g substrate, 40 ml incubation media and 10 ml
rumen fluid as described by Goering and Van Soest (1970). The incubation media (Table
4) was added to the flasks the night before the fermentation in order to hydrate the
substrates. The next morning, all flasks were placed in a water bath at 40°C, covered
with rubber stoppers and gassed continuously with C0,. All flasks were inoculated with
rumen fluid using an automatic syringe. Control rumen fluid samples were taken at the
beginning, midpoint and end of the inoculating process and stored at -10°C until
analyzed. Finally, two ml of a reducing solution (Table 3) were injected through the

inlet tube and 30 min later the isoacid mixture was added.

Table 4. Composition of the basal media.

 

Distilled water 500 ml
Trypticase“ 2.5 g
Micromineral solution 0.125 ml
Rumen buffer solution‘ 250 ml
Macromineral solution 250 ml
Resarzurin 1.25 ml

 

‘ This solution was prepared to provide different concentrations of NH, as required for
the low and high levels.

33

After 48 h of fermentation, 2 ml of the incubation mixture were taken for
analysis, and then 1 m1 of toluene was added to stop the fermentation and the flasks were

stored at 5°C. Zero time samples were taken.

Emufiheunalxsis

A sequential analysis of NDF, ADF, and ADL was conducted with the omission
of decahydronapthalene and NaSO, as modified by Robertson and Van Soest (1977). For
NDF, all of the incubation mixture was transferred to a 600 ml Berzelius beaker using
100 ml of neutral detergent solution. The mixture was refluxed for 1 hour and filtered
through a Gooch crucible, coarse porosity of 40 to 60 um. The crucibles were washed
two times with hot water and two times with acetone. Crucibles were air dried and then
dried overnight at 100°C, weight was determined at 100°C. For ADF, the sample in
the crucible was boiled in acid-detergent for 5 minutes and then transferred to a beaker
and refluxed for 55 nrinutes, filtered and dried as described for NDF. Crucibles with
ADP residue were treated with 72% sulfuric acid for 3 h, washed and filtered. Ashing
the residue at 500°C permitted the determination of lignin.

True dry matter digestibility was calculated as 100 minus the percent of neutral
detergent residue. Hemicellulose was calculated as NDF % - ADF% , and cellulose as

ADF% - lignin %.

34

5 ! . I . III I'

Determination of ruminal ammonia concentration was performed as described by
Chaney and Marbaeh (1962). A 5.00 pl aliquot of the incubation mixture was taken with
a Hamilton syringe and a Cheney adaptor and stored in a 4 ml plastic tube. One ml of
the phenol-nitroprusside and 1 ml of the sodium hydroxide-sodium hypochlorite solution
was added using a Micromedic systems automatic pipetting station, model 24004
(Micromedic Systems, Inc., Horsham, Pa.). The mixture was covered with aluminum
foil and incubated for 30 minutes at room temperature for color development.
Absorbances were read at 625 nm in a Stasar II spectrophotometer (Gilford Ins. Lab. ,
Burlington, Mass.). Ammonia standards were prepared from NH,C1 and ranged from

5-20 mg of ammonia/d1 of solution.

Badmamaujldeanalxsis

Ruminal hydrogen sulfide was determined according to manufacturers instructions
using a sulfide micro ion sensing electrode, Lazar model ISM-146 connected to a Lazar
model DPH digital pH meter (Lazar Res. Lab., Los Angeles, Ca.). A 250 pl aliquot of
the incubation mixture was combined with 250 pl antioxidant buffer (a mixture of 125
g sodium salieylate, 42.5 g NaOH, 32.5 g ascorbic acid and distilled water up to 500
ml). The standard HZS ranged from 0.01 to 100 ppm. The electrode was allowed to
stabilize in a 10 ppm standard sulfide solution for 30 minutes before being used, and for
10 minutes between recording sample readings. Rinsing of the electrode with distilled

water between samples was critical to obtain accurate readings. Potential readings

35

(millivolts) were plotted vs. sulfide concentrations on a logarithmic scale. Hydrogen

sulfide was expressed as mg/dl of incubation mixture.

 

VFA analysis was performed using a Hewlet Packard 5730 A gas liquid
chromatograph model 5730 A with flame ionization detector, a 7671 A automatic sampler
and a 3380 integrator. A glass column (30" x 1/4" x 4 mm ID) was packed with 60/80
Carbopaek C/0.3% Carbowax 20 M/0.1% H,PO,, lot # 146655 (Supelco cat. # 1-1825).
Nitrogen was the carrier gas at 50 ml/ min. The temperature program was 125°C for 4
minutes with a temperature increase of 8°C/ min for 5.63 min. The final temperature
was 170°C. Both, the injector and detector temperatures were 200°C.

One ml of each sample was centrifuged at 1000 RPM for 15 minutes, and a 10 pl
aliquot was used for analysis. Prior to the injection, the 1 ml samples were acidified
with 50 p1 of 88% formic acid (Baker 0128-01). The VFA standards contained 60, 20,
20, 2, 2, 2, 2, mM of C2, C3, C4, 1C4, 2MB, 1C5, and C5, respectively. Data output

was expressed as mM of the incubation mixture.

SI l' l' l l .
Overall significance of treatment effects was determined by AN OVA (Gill, 1978,

Vol. 1, 2 and 3) according to the model in equation 1. Differences among grasses and
legumes were determined by Bonferroni t-test and differences within the same specie

were determined by Scheffe (Gill, 1978a).

36
(1) Yam = F + at + 3, + (048), + 7x + (“7hr + (57),} + (037%,: + 5| + (06)..

+ (55%: + (75hr + (035% + (075M + (575% + (0575)h1d + E(ijk1)nr

where, p = the population mean
a, = species effect (fixed)
6,- = isoacid effect (fixed)
7, = nitrogen effect (fixed)
6. = sulfur effect (fixed)
(0:13),» (07);. (57);, (0:37),... (05%. (B5),» (75):» (ans)... (0175):.» (575%
and ((1575);,1d = interaction of the main effects (firted)

Em,“ = error term (random).

RESULTS

The effects of ammonia, S and isoacids on the volatile fatty acid concentrations
and true digestibility of tropical forages using an in vitro rumen fermentation procedure
are summarized in Table 5. Analysis of variance for in vitro true digestibility (IVTD)
showed a significant variation (P < .001) between all species and among grasses and
legumes (Table 6). Several interactions were significant. For species x NH,,
digestibility increased (P < .003) for all grasses as a result of increasing NH,. For
legumes, Gliricidia IVTD was not affected while Leucaena IVTD decreased when NH,
level was increased (Figure 4). Sulfur at high levels tended to decreased (P < .001)
IVTD of most of the forages (Figure 5). The interaction of isoacids x NH, (Figure 6),
showed that the addition of isoacids to treatments with high level of NH, decreased
(P < .032) digestibility of forages. When isoacids where added to treatments with low
level of NH,, IVTD increased.

The highest IVTD digestibility was found for Pennisetum and Panicum (64.61 and
64.89%) followed by Brachiaria and Digitaria (58.99 and 57.18%). Digestibility values
for Cenchrus and Cynadan were 53.40 and 53.85 % , respectively. Similar values were
obtained for these grasses by Kayongo-Male et a1. (1976). Stems from legumes had the
lowest digestibility, 44.52 and 29.18% for Gliricidia and Leucaena, respectively

(Table 5).

37

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Volatile fatty acid accumulation for the eight forage species studied is shown in
Figures 7, 8, and 9. The variation among species was significant (Table 7). Total VFA
production ranged from 44.07 to 71.05 mM (Table 5). Naga and Harmeyer (1975)
reported in vitro rumen total VFA production of 11.89 mMol - g" - h". Several in vivo
studies have been conducted with sheep. Bergman (1965) obtained a total VFA value of
109 mM of rumen fluid with dried grass diet. However, Leng and Brett (1966) found
a wide variation from 49.1 to 143.5 mM when sheep were fed four different diets
containing combinations of luceme, maize and Wheaten straw. Weller et al. (1969)
reported total VFA values of 100 to 136 mM in sheep grazing pastures. More recently,
Elliot et al. (1987) found a total VFA concentration of 48 mM.

In this study, the molar proportion of acetate was 70% which is similar to values
reported by Weller et al. (1969). However, the molar proportion of propionate (14 96)
was lower than values of 19 and 22% reported by Bergman (1965) and Weller et al.
(1969), respectively. It was observed that when acetate concentration was lower,
propionate was higher. Butyrate was fairly constant for all the species in the study
(Table 5).

A positive effect of isoacids on total VFA concentration was observed
(Figure 10). There was a variable effect of the interaction of species x NH, on acetate

production as illustrated in Figure 11.

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It was found that high NH3 decreased (P < .063) acetate production from

Brachiaria, Cenchrus, and Panicum while an increase in NH3 increased acetate
production from Cynodom, Digitaria, Gliricidia, and Pennisetum. 0n the other hand,
there was a negative effect (P < .061) of isoacids on propionate concentration when NH,
level was high. However, a positive effect of isoacids on propionate production was
found with low levels of NH3 (Figure 12). For the interaction of isoacids x S on total
isoacids, the high level of isoacids increased (P < .046) the total concentration of
isoacids for both low and high levels of 8 (Figure 13).

Isoacid concentrations after a 48 h in vitro rumen fermentation of tropical forages
are presented in Table 8. Concentrations of C4 and C5 fatty acids were found to vary
among species (P < .061). Isovalerate ranged from 0.75 to 1.05 mM. Valerate
concentration ranged from 1.19 to 2.28 mM. The values for 2-methylbutyrate ranged
from 0.61 to 0.92 mM accounting for differences between species. However, isobutyrate
concentration was similar for all species studied (Table 8). Hefner (1985) reported
ruminal molar proportions of isobutyrate and 2-metylbutyrate + isovalerate to be 6.3 and
1.5 molar % of total VFA when lambs were fed a corn diet supplemented with urea and
isoacids. Umunna et al. (1975) found that addition of isobutyrate and isovalerate to high
roughage diets fed to lambs did not affect dry matter digestibility. Similar results were

obtained by Hemsley and Moir (1963), Cline et al. (1966) and Hume (1970a and b).

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Ammonia and sulfide concentrations after a 48 h in vitro rumen fermentation of
tropical forages are presented in Table 8. Significant differences (P < .05) in NH3
concentration were observed between species. Values ranged from 17.30 to 26.94 mg
NH3/dl for Gliricidia and Cynodon, respectively. The use of Trypticase in the incubation
media explains the high levels of NH, found after the 48 h incubation. Sulfur
concentrations ranged from 0.14 to 0.22 mg H28/dl for Pennisetum and Panicum and
Brachiaria, respectively. However, these sulfide levels were at the limits of detection
and are not considered significant.

The effects of NH,, S and isoacids on the in vitro rumen fermentation of
Brachiaria decumbens are summarized in Table 9. It was observed that IVTD (%)
ranged from 53.50 (LHH) to 62.10 (LLH), although values were not statistically
different. However, it was noticed that high levels of S (LI-IL) tended to decrease IVTD
and the addition of isoacids (LHH) further decreased digestibility. The addition of high
levels of NH3 (HHH) tended to restore digestibility. A similar trend was observed for
all other species. However, Quispe et al. (1991) found a positive effect of isoacids and
S on acetate production when sheep were fed pineapple tops. The treatments for
Brachiaria were not significantly different from each other for all the parameters studied.
Lignin digestibility values were negative suggesting this was probably due to precipitation

by phenolic compounds.

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61

The effects of the three factors (NH,, S and Isoacids) on the in vitro rumen
fermentation of Cenchrus ciliaris are summarized in Table 10. IVTD values ranged from
50.53 (LLL) to 56.83% (HLL) which are close to in viva values reported by Minson and
Bray (1986) of 58.7% but different from that (66.7%) reported by Yerena et a1. (1978).
The treatment HLL showed the highest NH3 concentration (29.10 mg NIL/d1) which
significantly decreased (21.92 mg NH3/dl) when S was added (treatment HHL). Results
for the in vitro rumen fermentation of Gynodom dactylon are presented in Table 11.
Some of the treatments were statistically significant for IVTD. The highest values were
found for treatments with high level of NH,, 57.22, 56.68, and 56.04% for HLH, HHL,
and HLL, respectively. Total VFA and isoacid concentration did not differ among
treatments.

For Digitan'a decumbens, IVTD ( %) was not different between treatments (Table
12). Once again the highest IVTD value was found for high NH, level on treatment
HLL (62.71%) and the lowest (52.83%) when high S was added (HHL). Hunter and
Siebert (1986) found an apparent organic matter digestibility of 59% with grazing cattle.
It was observed that the decreased IVTD due to S supplementation was corrected with
the addition of isoacids as shown for treatment HHH (58.39%). Total VFA was not
affected by the treatments. The effects of NH3, S and isoacids on the in vitro rumen
fermentation of Gliricidia sepium are summarized in Table 13. In vitro true digestibility
did not differ among treatments and ranged from 41.64% (HLH) to 46.09% (HLL).
Acetate production was higher (P < .05) for treatment HLH (56.36 mM) than from all

other treatments which averaged 23.57 mM.

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65

 

 

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66
For Leucaena leucocephala, IVTD values were lower than all other species and

differed slightly between treatments. Values obtained ranged from 25.76% (HHH) to
34.82% (LLH). Vadiveloo (1989) and Yerena et al. (1978) found dry matter digestibility
values of 5 8.5 and 59.7% when Leucaena leaves were fed to goats and lambs,
respectively. Treatment with addition of high 8 and isoacids (LI-1H) showed the highest
isoacid concentration which was 4.63 mM (Table 14). IVTD values of Panicum
maximum were found to differ (P < .05) with treatments HLH and LHL showing the
highest (67.35%) and lowest (62.61%) values, respectively. However, IVTD (96) value
obtained for this species was the highest (64.89%) of all species studied. Treatment with
high NH,, S and isoacids (HHH) gave the highest value for total isoacid concentration
(5.72 mM). However, when S and isoacids were low (HLL), it decreased to 3.22 mM
(Table 15). Results for Pennisenun purpureum showed no significant differences among
treatments for any of the parameters studied (Table 16). A value of 64.61 % was found
for IVTD.

Apparently, S exerted a toxic effect on the digestibility of Panicum maximum
(Table 15). This differs from previous studies with herbage diets . where increased
digestion of organic matter or dry matter and ADF (in viva) were found as a result of the
addition of S to low S herbage (Weston et al., 1988; Rees et al., 1974; Guardiola et al.,
1983; Bray and Hemsley, 1969; Kennedy and Siebert, 1972). On the other hand,
Kennedy (1974) reported that digestion of tropical spear grass was not limited by S intake
although VFA concentration was increased by sulphate supplementation accompanied by

a small decrease in propionic and isovaleric acids.

DISCUSSION

There are several interesting results from this study. First, IVTD was highly
variable among species. By increasing NH,, IVTD was increased for all grasses. This
suggests that NH, may be limiting in the early stages of fermentation. At the end of the
48 h fermentation NH, levels were about the same for all treatments within a grass
species. This may be due to the fermentation of trypticase which would result in the
levels of ammonia observed. Isoacids are produced from trypticase. However, when
the experimental treatments included high levels of isoacids, the levels of total and
individual isoacids were about doubled compared to low isoacids treatments at 48 h for
most of the forage species. This shows that an increase in isoacid levels was
accomplished by adding isoacids to the fermentation media.

The high NH, level accompanied by high isoacids decreased IVTD. However,
when the high level of isoacid was added to treatments with low levels of NH,, IVTD
was increased. It may be that the first limiting factor in the fermentation was NH, with
isoacids a second limiting factor because adding isoacids to low NH, treatments increased
digestibility. But adding isoacids when requirements for NH, were met decreased IVTD.
It is recognized that the explanation of treatment effects on IVTD is confounded by the

addition of trypticase to the media. Since high isoacids along with low levels of NH,

67

68

increased IVTD, it may be that these effects occurred early in the incubation before
trypticase was extensively fermented. Cline et al. (1966) found that cellulose and dry
matter digestibility was improved by the addition of isoacids in lambs fed a purified diet
containing 39% cellulose and urea. Ruminal NH, levels were lower than in the present
study. Similar results were obtained in vitro by Cummins and Papas (1985). However,
Hefner et al. (1985) observed that when urea and isoacids were both supplemented the
tendency was to decrease the extent of digestion concluding that isoacid deficiency is not
the first limiting factor affecting digestion in ruminants.

Variation in IVTD among all forages was basically due to differences in the fiber
fractions. Grasses averaged 41.12% NDF, 23.04% ADF and 4.28% lignin while values
for legumes were 63.87%, 49.47% and 14.62% for NDF, ADF and lignin, respectively.
This explains the large differences in digestibility between legumes and grasses. Within
grasses Panicum (35.5% NDF, 20.62% ADF) showed the highest digestibility value
(64.89 %) and Cenchrus (45.37% NDF, 25.87% ADF) had the lowest digestibility
(53.40%). ’

Concerning the digestibility of the different fiber fractions, it was observed that
Pennisetum (Table 16) had the highest values for ADFD, CWD, HEMD and CELD.
Digestibility of those fractions were similar within all other grasses (Table 9, 11, 12,
15). However, ADFD, CWD, HEMD and CELD values for Cenchrus were slightly
lower (Table 10). It was clear that digestibility of hemicellulose was closely related to
that of cellulose for all forages and negatively correlated with lignin. The legumes,

Gliricidia and Leucaena were very high in lignin and, therefore, digestibility of cellulose

69

and hemicellulose was less than the grasses. There was no significant difference between
treatments in the digestibility of the fiber fractions of Brachiaria, Cenchrus, Digitaria,
Gliricidia and Pennisetum. However, there were slight differences between treatments
on fiber fractions for Cynodon, Leucaena and Panicum.

Volatile fatty acid accumulation varied among species perhaps due to differences
in fiber content. When acetate concentration was lower, propionate was higher as
expected. In vitro true digestibility of forages was more positively correlated with
propionate than with acetate for Digitaria, Gliricidia, Leucaena, Panicum and
Pennisetum. However, the opposite trend was observed for Brachiaria, Cenchrus and
Cynodon. For Brachiaria, Cenchms and Panicum high levels of NH, decreased acetate
concentration but increased IVTD. However, for Gynodon, Digitaria, Gliricidia and
Pennisetum the increase in NH, accounted for an increase in propionate concentration
which increased IVTD. The levels of VFA in the 10 ml of rumen fluid used to inoculate
the media were low (e. g. 40 mM acetate) and were not subtracted from the 48 h VFA
values because they would not affect treatment differences.

Isoacid addition increased total VFA accumulation and for some species increased
acetate concentration (Brachiaria, Digitaria and Gliricidia). Treatments with high levels
of NH, and isoacids decreased propionate concentration. This decrease in propionate
could be a possible explanation for the decrease in IVTD discussed above. NH,
concentration differed between species probably due to differences in available protein.

V 0f major interest was the S inhibition of IVTD. This observation was

unexpected. High levels of S were found to decrease IVTD. When high S and isoacids

70
were combined, IVTD further decreased. However, adding high NH, to a system high

in S and isoacids partially prevented the inhibitory effect of S and isoacids. Bird (1972b)
reported that sulfate supplementation without urea decreased N balance and depressed
digestion of feed in ruminants. As discussed above, adding NH, to the system increased
digestibility. This may explain why NH, tends to alleviate the inhibitory effect of S and
isoacids. However, this does not explain why there is a negative synergistic effect
between S and isoacids on IVTD. Since the trend was for S to increase total isoacid
levels at 48 h, it may be that S stimulated trypticase fermentation at the expense of fiber
digestion.

Kennedy (1974) found that S supplementation inhibited propionate fermentation
in his experiment which could explain a decrease in digestibility and Morrison et al.
(1990) observed that S increased acetate but lowered propionate. However, in our study
there is no evidence that S affected propionate concentration.

For Cenchrus, treatments with low S increased NH, concentration but treatments
with high S decreased digestibility and affected NH, levels. The highest digestibility for
Digitaria was found when the S level was low. The same pattern was found for most
species with the exception of Permisetum. This is evidence that S inhibited the
fermentation. Another possible explanation is that S level and reducing potential may
have been confounded. Sulfide acts as a reducing agent. Treatments with high levels
of S had three times more sulfide added than did low S treatments. There may have
been significant differences in reducing potential early in the incubation period that

affected both trypticase fermentation and IVTD.

SUMMARY

There were three two-way interactions, species x NH,, species x S and isoacids

x NH, which affected IVTD. High NH, increased IVTD for all forages except

the legumes.

High levels of S tended to decrease IVTD for all forages, except Pennisetum.

The addition of isoacids (15 mg/dl) to low NH, increased digestibility, but

decreased IVTD when NH, was high. Also, addition of isoacids increased total

VFA accumulation.

71

CONCLUSION

In the 48 h in vitro ruminal fermentation end point assay using the standard
trypticase addition to the media, it is concluded that to prevent inhibition of IVTD, S
levels must be less than 6 mg/dl. Also, added NH, levels of 10 mg/dl improved
digestibility. IVTD can also be increased by adding isoacids at 15 mg/dl along with NH, -

at 5 mg/dl.

72

APPENDIX

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Table 26. Fiber fraction (96) of tropical forages after a 48 h in vitro rumen fermentation.

 

 

 

 

 

NDF ADF LIG
(95) (95) (95)
Brachian'a decumbens 41.25 22.38 4.25
Cenchrus ciliaris 45.37 25.87 4.00
Cynodon dactylon 45.75 23.50 5.34
Digitaria decumbens 42.87 25.50 4.75
Gliricidia sepium 56.00 43.75 12.25
Leucaena leucocephala 71.75 55.25 17.00
Panicum maximum 35.50 20.62 4.00
Pemzisetum purpureum 36.00 20.37 3.37
Table 27. Dry matter and crude protein of tropical forages.
Dry matter Crude Protein
(95) (95)
Brachiaria decumbens 95.61 6.80
Cenchrus ciliaris 96.62 5.06
Cynodon dactylon 96.32 6.20
Digitaria decumbens 95 .64 7.80
Gliricidia sepium 94.28
Leucaena leucocephala 94.62 6.00
Panicum maximum 96.38 5 .28
Pennisemn purpureum 92.73 7.70

 

Table 28. Hemicellulose and cellulose fractions (96) of tropical forages after a 48 h in

vitro rumen fermentation.

85

 

 

Hemicellulose Cellulose
(95) (96)
Brachiaria decumbens 18.28 17.46
Cenchms ciliaris 19.62 21.99
Cynodon dactylon 22.38 18.01
Digitaria decumbens 17.06 21.14
Gliricidia sepium 12.42 27.34
Leucaena leucocephala 16.63 38.39
Panicum maximum 14.90 16.41
Pennisetum pulpureum 15.63 16.98

 

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