Animal Feed Science and Technology

93 (2001) 115

In vitro rumen fermentation and gas production:

inuence of yellow grease, tallow, corn oil
and their potassium soaps
G. Getachew, E.J. DePeters*, P.H. Robinson, S.J. Taylor
Department of Animal Science, University of California, Davis, CA 95616-8521, USA
Received 29 January 2001; received in revised form 29 May 2001; accepted 13 June 2001

Abstract

The effect of addition of fat on in vitro gas production, volatile fatty acid (VFA) production, in
vitro true digestibility (IVTD), and ammonia-N concentration was assessed by incubation of a
simulated total mixed ration in buffered rumen uid using an in vitro gas technique. Fat sources
were corn oil (CO), tallow (TL), yellow grease (YG), and potassium soaps (K-soaps) of CO, YG,
and TL. Addition of YG increased (P < 0:001) in vitro gas production while TL had no effect.
Neither YG nor TL affected IVTD and total VFA production. Addition of either YG or TL
decreased acetate production and increased propionate production. Addition of CO increased in
vitro gas production, but had no effect on IVTD and total VFA production. However, CO decreased
acetate and increased propionate production with a concomitant decrease in the acetate to
propionate ratio. Addition of K-soaps of CO, TL and YG depressed in vitro gas production, IVTD,
VFA and ammonia-N concentrations. K-soaps also caused a marked decrease in acetate and
increase in propionate production. The absence of negative effects on rumen fermentation
parameters in response to the inclusion of fat in the form of triglycerides to an in vitro system at up
to 25% DM suggests that triglycerides have much less effect on rumen fermentation parameters
than corresponding free fatty acids. In contrast, the K-soaps of each respective fat had detrimental
effects on in vitro rumen fermentations. # 2001 Elsevier Science B.V. All rights reserved.

Keywords: Gas production; Fats; In vitro

1. Introduction

Inclusion of fats and oils in rations of lactating dairy cows increases the caloric density
and increases energy intake to support higher milk yield during early lactation when cows

*
Corresponding author. Tel.: 1-916-752-0175; fax: 1-916-752-0175.
E-mail address: ejdepeters@ucdavis.edu (E.J. DePeters).

0377-8401/01/$ see front matter # 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 1 ) 0 0 2 6 4 - 4
2 G. Getachew et al. / Animal Feed Science and Technology 93 (2001) 115

are often in negative energy balance. There are conicting reports on the effects of added
fat on rumen fermentation. Some workers have reported that added fat had no adverse
effects on ber digestibility (Palmquist and Conrad, 1978; Hussein et al., 1995; Bateman
and Jenkins, 1998) while others reported reductions in digestibility of ber (Brooks et al.,
1954; Pantoja et al., 1994; Eastridge and Firkins, 2000), and organic matter (OM;
Ikwuegbu and Sutton, 1982). There have been several hypotheses proposed to describe
how added dietary fat affects microbial activity in the rumen. It has been suggested that
dietary fats may coat ber and interfere with microbial attachment thus leading to
depressed ber digestibility (Devendra and Lewis, 1974). Stewart (1977) observed a
depression in cotton ber degradation when the cotton yarn had been soaked in either
tallow or fatty acids. In contrast, Demeyer and Van Nevel (1995) reported no difference in
cellulose degradation when it was coated, or uncoated, with soybean oil hydrolysate,
leading these researchers to conclude that inhibitory effects of fat were not a consequence
of fatty acid adsorption to ber. Inclusion of fat in the diet caused a linear depression in
intake and ber digestibility (Orskov et al., 1978). Dietary fat may modify the ruminal
microbial population, which is responsible for cellulose digestion. Henderson (1973)
reported that fatty acids inhibited growth of a cellulolytic organism in vitro, but had no
effect on other organisms that produced propionate. Fats and fatty acids were shown to alter
the proportion of individual volatile fatty acids (VFAs) in an in vitro system (Chalupa et al.,
1984, 1986; Jenkins, 1987). The reduced availability of calcium needed for microbial
function could be a mechanism by which dietary fat inhibited microbial fermentation
(Jenkins, 1993). Galbraith et al. (1971) using pure culture techniques and El Hag and Miller
(1972) employing in vivo experiment demonstrated that individual fatty acids inhibited
microbial growth, but inclusion of calcium reversed the inhibitory effect.
In vivo studies conducted to assess the effect of fats on production and/or rumen
fermentation parameters have failed to yield consistent results. This could be due to a
variety of factors that determine the activity of fats on rumen microbes. Such factors might
include the source of fat, degree of saturation, fatty acid chain length, whether the fat was
added as a triglyceride or as a free fatty acid, as well as other undescribed characteristics of
fats, such as rate and extent of hydrolysis. Short chain fatty acids have been shown to cause
greater depression of ber digestibility than long-chain fatty acids (Steele and Moore,
1968). Unsaturated oils had a greater negative effect than saturated fats (Macleod and
Buchanan-Smith, 1972; Jenkins, 1994), and free fatty acids caused a larger negative effect
than the corresponding triglycerides (Macleod and Buchanan-Smith, 1972; Bateman and
Jenkins, 1998). Machmueller et al. (1998) reported that rumen protected fat (Efeco, UFAG,
Switzerland), as well as whole rapeseed, had less effect on the rumen fermentation pattern
and methane production than coconut oil. However, Bateman and Jenkins (1998), using
soybean oil (8% of dietary DM) concluded that large amounts of unprotected fat could be
added to diets without depressing digestibility. Bateman et al. (1996) reported a depressive
effect of added tallow on intake of a low ber diet, but not a high ber diet, suggesting that
high ber in the diet promotes conditions for rapid growth of microbes that hydrolyze and
hydrogenate dietary fat.
The studies reported here were designed to determine effects of several levels of added
fat to a simulated total mixed ration on in vitro gas production, in vitro true digestibility
(IVTD) of dry matter, and VFA production. In addition, comparison of the inuence of
 G. Getachew et al. / Animal Feed Science and Technology 93 (2001) 115 3

added fat in the form of triglycerides (i.e. yellow grease and tallow which contain about
95% fatty acids in the form of triglycerides; Avila et al., 2000) and in the form of potassium
soaps (K-soaps) on in vitro fermentation.

2. Materials and methods

2.1. Preparation of diet and potassium soaps

A simulated total mixed ration (STMR) was prepared using beet pulp (7.1%), soybean
meal (6.9%), barley grain (18%), corn grain (18%), and alfalfa hay (50%). The STMR was
ground to pass a 1 mm screen in a Wiley mill. The diet contained 94.4% OM, 18.7% CP,
1.7% EE, and 19.0% ADF, and 32.0% NDF (DM basis). Corn oil (CO) was obtained from
Safeway (Safeway Inc., Pleasanton, CA), and yellow grease (YG) and tallow (TL) were
supplied by Foster Commodities (Foster Commodities, Fresno, CA, USA). Potassium
soaps (K-soaps) of TL (TL-soap), YG (YG-soap), and CO (CO-soap) were prepared
following the principle described in Gunstone et al. (1994) with advice from Palmquist
(personal communication). Briey, about 500 ml of 100% ethanol and 30 ml of 50% KOH
were added into a 1 l beaker containing 25 g of fat or oil. The TL and YG were heated
before weighing into the beaker. The beaker was covered with a watch glass and placed on
a steam table overnight. The cover was removed until the ethanol was evaporated to about
100 ml. About 200 ml of de-ionized water were added and the solution was stirred gently
with a glass rod. The content of the beaker was transferred into a separatory funnel and
about 200 ml of hexane was added. The solution was shaken by hand for about 3 min and
allowed to settle. The bottom layer was transferred into a plastic container, freeze-dried,
and nely crushed with a pestle in a mortar. The powder was stored refrigerated (58C) until
used for in vitro incubation.

2.2. In vitro gas production

2.2.1. Experiment 1
In vitro incubation was performed using 40 ml of buffered rumen uid according to the
method of Menke et al. (1979) as described in Makkar et al. (1995). Approximately 500 mg
of STMR were incubated alone (control, no added fat) or with the equivalent of 5, 10, 15,
20, and 25% added fatty acids as TL or YG. The STMR and fats were weighed on a
weighing boat with a removable glass rod and placed in 100 ml graduated glass syringes.
The piston was lubricated with Vaseline and inserted into the syringes. Buffer mineral
solution (Makkar et al., 1995) was prepared and placed in a water bath at 398C under
continuous ushing with CO2. Rumen uid was collected after the morning feeding from
two rumen stulated, nonlactating, nonpregnant Holstein cows (about 650 kg body weight)
fed oat hay in the long form in two equal feedings (7.00 and 18.00 h) daily. Rumen uid
was pumped with a manually operated air pump from the rumen into pre-warmed thermos
asks. The rumen uid from the two cows was mixed and ltered through four layers of
cheesecloth and ushed with CO2. The well mixed and CO2 ushed rumen uid was added
to the buffered mineral solution (1:3 v/v), which was maintained in a water bath at 398C,
4 G. Getachew et al. / Animal Feed Science and Technology 93 (2001) 115

and mixed. Buffered rumen uid (40 ml) was pipetted into each syringe containing ground
STMR as the substrate and the various fat sources. The syringes were immediately placed
in a water bath at 398C. Each incubation was performed in quadruplicate. Gas production
was recorded up to 24 h and the syringes were shaken every hour for the rst 8 h of
incubation. The total in vitro gas and VFA produced were corrected for blank incubations
(i.e. no substrate).

2.2.2. Experiment 2
Procedures similar to experiment 1 were used. Approximately 500 mg of STMR was
incubated alone (control, no added fat) or with the equivalent of 5, 10, 15, 20, and 25% of
added fatty acids from CO or potassium soaps (K-CO). Incubations were performed in
quadruplicate. At the end of the 24 h incubation period, the contents of two of the four
syringes were centrifuged at 11,000  g for 20 min. and the supernatant was used for VFA
and ammonia-N analyses. For VFA determination, 10 ml of syringe contents were pipetted
into a plastic tube and centrifuged at 11,000  g. Supernatant (5 ml) was then pipetted into
a plastic tube containing 1 ml of 25% meta-phosphoric acid solution. Each tube was
centrifuged and 0.1 ml of supernatant was pipetted into gas chromatography (GC) vials
containing 0.9 ml of internal standard (i.e. 3-methylvaleric acid) and sealed. For IVTD
determination, the contents of the remaining two syringes were each transferred to 600 ml
beakers, reuxed in neutral detergent (ND) solution for 1 h, and ltered through a tared
crucible. The NDF residue measured from blank incubation was negligible and therefore
blank correction was not necessary. The IVTD was calculated as the weight of substrate
incubated minus the weight of residue after ND treatment, and expressed as a percent of
substrate incubated. Each in vitro gas production 24 h run was replicated once.

2.2.3. Experiment 3
Procedures similar to experiments 1 and 2 were used. Approximately 500 mg of STMR
were incubated alone (control, no added fat) or incubated with the equivalent of 5, 10, 15,
20, and 25% of added fatty acids as YG or K-soaps of YG in two runs. The TL and K-soaps
of TL were used in the following two runs. Incubations were performed in quadruplicate
with other procedures as described for experiment 2.

2.3. Chemical analysis

The DM, OM, and ether extract (EE) were determined according to AOAC (1990).
Neutral detergent ber (NDF) and acid detergent ber (ADF) were analyzed following the
method of Van Soest et al. (1991). Fatty acid contents of YG, TL, CO and their K-soaps
were determined using the method of Sukhija and Palmquist (1998) but modied according
to Joy et al. (1997) and Crocker et al. (1998). The VFA were analyzed using a Hewlett-
Packard 5890 Capillary Gas chromatograph tted with a J&W OV 351 capillary column
(FFAP phase), 3 m  0:25 mm. The initial temperature of the column was 1008C, set at a
rate of 48C/min until a nal temperature of 1408C was reached with a total running time of
30 min. Injector and detector temperatures were 220 and 2308C, respectively, with carrier
gas (H2) head pressure of 17.1 psi and the split vent ow rate of 100 ml/min. Ammonia-N
was determined with an autoanalyzer procedure (Chaney and Marbach, 1962).
 G. Getachew et al. / Animal Feed Science and Technology 93 (2001) 115 5

2.4. Statistical analysis

The effect of YG, TL, CO and K-soaps of YG, TL and CO on in vitro gas production,
IVTD, VFA and ammonia-N was assessed using linear regression procedures (SAS, 1982).

3. Results and discussion

3.1. The effect of YG or TL on in vitro fermentation of feed

Fat sources varied in their fatty acid composition as expected (Table 1). The TL was a
more saturated fat source than YG.
Addition of YG increased (P < 0:001) in vitro gas production 0.35 ml gas/g DM (per
percent) increase in fatty acids in YG (Table 2). TL did not affect gas production. Neither
YG nor TL had an effect on IVTD. This observation is consistent with the ndings of
Bateman and Jenkins (1998) who observed no effect on DM or ber digestibility by
supplementation of soybean oil. In contrast, Pantoja et al. (1994) reported a decline in ber
digestibility as the level of unsaturation of the fat in the diet of lactating cows was
increased. Total VFA production was not affected by addition of either YG or TL but

Table 1
Fatty acid (DM, per percent) composition of yellow grease (YG), tallow (TL), corn oil (CO), and potassium
soaps of YG, TL and CO

Components TL CO YG Potassium soaps of

TL CO YG

C10 0.049 0.036 0.026

C12 0.081 0.076 0.051 0.052
C14 2.259 0.030 0.975 1.507 0.693
C14:1 trans 0.158 0.099
C14:1 cis 0.359 0.163 0.122 0.024
C15 0.391 0.157 0.237
C16 21.370 9.215 15.879 14.093 7.605 11.153
C16:1 trans 0.273 0.074 0.209 0.058
C16:1 cis 2.714 0.085 2.247 1.804 0.078 1.593
C17 1.012 0.065 0.379 0.677 0.056 0.281
C17:1 trans 0.091 0.281
C18 16.157 1.733 9.035 10.743 1.445 6.419
C18:1 trans-9 0.275 1.523 0.174 1.110
C18:1 trans-11 1.103 2.095 0.719 1.412
C18:1 cis-9 and -10 27.914 23.928 27.697 18.614 19.724 19.444
C18:2 3.506 48.142 10.811 2.267 39.020 7.699
C18:3 0.414 0.861 0.698 0.242 0.676 0.505
C18:2 cis-9 and trans-11 0.218 0.131 0.038
C20:4 0.100 0.091 0.050 0.10
Others 0.846 1.371 6.45 3.754 2.412 8.727
Total fatty acids 79.29 85.43 78.35 55.81 71.04 59.31
6 G. Getachew et al. / Animal Feed Science and Technology 93 (2001) 115

Table 2
Effect of level of YG and TL on in vitro gas production in vitro true digestibility (IVTD) and volatile fatty acid
(VFA) production on incubation of mixed ration in buffered rumen fluid (experiment 1)

Parameters Intercepta Slopeb P-valuec S.E.

YG TL YG TL

Gas production (ml/24 h/g DM) 262.2 0.35 0.06 <0.001 0.46 4.6
IVTD (DM, per percent) 83.3 0.048 0.012 0.18 0.74 1.27
VFA (mmol/g DM)
Total 5.65 0.005 0.008 0.44 0.23 0.22
Acetate 3.43 0.012 0.013 0.02 0.01 0.18
Propionate 1.47 0.010 0.007 <0.001 <0.001 0.05
Butyrate 0.55 0.001 0.0005 0.60 0.65 0.04
iso-Valerate 0.08 0.0007 0.001 0.008 <0.001 0.01
Valerate 0.117 0.0 0.0 0.98 0.74 0.01
Acetate:propionate 2.32 0.019 0.17 <0.001 <0.001 0.15
a
The intercept is the fitted value at zero level of fatty acids either from YG or TL.
b
The slope is the unit of change per percent increase in fatty acids per gram of DM feed.
c
The P-value indicates the probability that the slope differs from zero.

addition of either YG or TL decreased (P < 0:001) acetate production and increased

(P < 0:001) propionate production. Consequently, the acetate to propionate ratio was
reduced (P < 0:001) by addition of either YG or TL to the incubation. Neither YG nor TL
affected production of butyrate and valerate.
Chalupa et al. (1984) reported that triglyceride additions of palmitate, stearate or oleate
to in vitro incubations did not affect total VFA production, proportions of acetate and
propionate, and the acetate to propionate ratio compared to the control. Similarly, TL fatty
acids in the triglyceride form added at 10, 15, or 20% levels to in vitro incubations did not
affect total VFA production, production of acetate and propionate, and the acetate to
propionate ratio compared to the control (Chalupa et al., 1984). This is consistent with
Jenkins (1987), who reported that addition of blended animalvegetable fat at up to 10% of
DM in in vitro incubations had little effect on fermentation. Machmueller et al. (1998),
using a rumen simulation technique (RUSITEC), evaluated the impact of ``fatty feeds''
including coconut oil and whole crushed rapeseed, sunower seed, and linseed meal on
in vitro rumen fermentation. Although the inclusion of fatty feeds, with the exception of
rapeseed, reduced the quantity of OM fermented, total production of VFA was not
affected and only the proportions of individual VFA were affected by addition of fatty
feeds. The majority of the in vitro rumen fermentation studies have not demonstrated
that addition of fats in the triglyceride form depressed fermentation. Although changes
in proportion, of individual VFA, and methane production, were previously reported,
total VFA production was largely unaffected by fat addition, which is in close agreement
with the results of present study. The reduction in the acetate to propionate ratio in the
present study was also consistent with in vivo (Kowalczyk et al., 1977; Ikwuegbu and
Sutton, 1982; Boggs et al., 1987) and in vitro (Czerkawski and Clapperton, 1984;
Jenkins, 1987) results.
 G. Getachew et al. / Animal Feed Science and Technology 93 (2001) 115 7

A reduction in the acetate to propionate ratio often improves the efciency of feed
utilization, since relatively higher propionate production is associated with less methane
production and so less loss of energy in the form of gas. Methane production was reported
to be lower in fat supplemented diets compared with the control (Machmueller et al., 1998).
The reason for the methane suppressing effects of fats could be through a direct inuence
on the rumen methanogenic microbes.
The threshold at which added fats exert a negative effect on rumen fermentation in vivo
appears to vary depending on the basal diet, source of fat, and its level of saturation. For
instance, Jenkins (1994) concluded that ruminant diets containing more than 24% added
fat are likely to depress ber digestion in the rumen, but this has not been supported by
ndings of others (Hussein et al., 1995; Plascencia et al., 1999; Firkins et al., 1990; Doreau
et al., 1991) where no adverse effects were observed on ber digestion at higher levels of
added fat. Based on in vivo work, Firkins et al. (1990) reported no difference in ber
digestibility at 5% supplementation of fat and at 3% dietary supplementation of soybean
oil, feed efciency, VFA, and average daily gain were increased (Whitney et al., 2000).
Chalupa et al. (1986) found that addition of 10% long chain fatty acids did not affect total
VFA production but decreased acetate and increased propionate proportions. However,
these authors recommended a maximum of 68% fat supplementation. Ikwuegbu and
Sutton (1982) reported an increase of total VFA production when linseed oil was
supplemented. Neither total nor ruminal digestibility of ber or organic matter was
affected by supplementation of 10% tallow or rapeseed oil (Doreau et al., 1991). An in
sacco study has indicated that at short incubation (i.e. 12 h), fat depressed DM disap-
pearance and ammonia concentration but this did not occur at longer incubations (i.e. 48 h;
Doreau et al., 1991).

3.2. The effect of potassium soaps of CO on in vitro fermentation of feed

Since TG and TL had no effect on in vitro fermentation in experiment 1, and Jenkins

(1987) had previously demonstrated that CO did affect in vitro rumen fermentation, CO
was used in experiment 2. Since the fatty acid composition of CO was more unsaturated
than both TL and YG (Table 1) expectations were that CO might suppress rumen
fermentation. In fact, addition of CO increased (P < 0:001) in vitro gas production
0.38 ml gas/g DM (per percent) increase in CO (Table 3) and there was no effect of
CO on IVTD and total production of VFA. However, CO decreased acetate (P < 0:05) and
increased propionate (P < 0:01) production with a concomitant decrease in the acetate to
propionate ratio (P < 0:01). In the earlier work of Jenkins (1987), CO decreased the
acetate to propionate ratio and concentration of propionate while in another trial CO
reduced NDF digestibility and total VFA production. In contrast to CO (Table 3, Fig. 1),
K-CO depressed (P < 0:001) gas production 3.01 ml gas/g DM (per percent) increase
in fatty acids in the soap. Mean IVTD and total VFA production were not affected by
addition of K-CO, although there was a decrease (P < 0:001) in acetate and an increase
(P < 0:001) in propionate production resulting in a reduced (P < 0:001) acetate to
propionate ratio.
Jenkins (1993) suggested that unsaturated fatty acids have more potent anti-microbial
effects and so promote greater inhibition of ruminal fermentation. Even though CO was
8 G. Getachew et al. / Animal Feed Science and Technology 93 (2001) 115

Table 3
Effect of level of CO and potassium soaps of CO in vitro gas production in vitro true digestibility (IVTD) and
volatile fatty acid (VFA) production on incubation of mixed ration in buffered rumen fluid (experiment 2)

Parameters Intercepta Slopeb P-valuec S.E.

CO CO-soap CO CO-soap

Gas production (ml/24 h/g DM) 251.9 0.38 3.01 <0.001 <0.001 4.6
IVTD (DM, per percent) 82.3 0.044 0.05 0.25 0.26 1.65
VFA (mmol/g DM)
Total 5.69 0.0 0.004 0.46 0.52 0.23
Acetate 3.25 0.006 0.034 0.04 <0.001 0.13
Propionate 1.77 0.01 0.05 0.01 <0.001 0.17
Butyrate 0.48 0.0 0.01 0.64 <0.001 0.03
iso-Valerate 0.067 0.0 0.003 0.28 <0.001 0.02
Valerate 0.115 0.0 0.004 0.54 <0.001 0.01
Acetate:propionate 1.8 0.01 0.04 <0.01 <0.001 0.12
Ammonia-N (mg/l) 185.3 0.7 2.02 0.30 <0.05 28.9
a
The intercept is the fitted value at zero level of fatty acids either from CO or CO-soaps.
b
The slope is the unit of change per percent increase in fatty acids per gram of DM feed.
c
The P-value indicates the probability that the slope differs from zero.

Fig. 1. The effect of level of fatty acids in CO and CO-soap on in vitro gas production.
 G. Getachew et al. / Animal Feed Science and Technology 93 (2001) 115 9

more unsaturated in fatty acid composition than the TL and YG used in experiment 1, CO
did not inuence IVTD and total VFA production. However, the addition of CO-soaps,
which dissociate in the rumen, negatively affected gas production. Chalupa et al. (1984)
reported that addition of TL fatty acids as calcium salts to in vitro incubations did not affect
total VFA production. Calcium salts are reported to have little dissociation in the rumen
(Chalupa et al., 1984). In contrast, the addition of TL fatty acids in the free form depressed
total VFA production compared to the control. This response agrees with the current
observations for K-soaps of CO.

3.3. The effect of yellow grease, tallow and potassium soaps of yellow grease
and tallow on in vitro fermentation of feed

Inclusion of YG increased (P < 0:01) in vitro gas production but had no effect on
IVTD or VFA production (Table 4). Addition of K-YG, in contrast depressed in vitro
gas production (P < 0:001), IVTD (P < 0:001) and total VFA production (P < 0:001).
K-YG resulted in a marked decrease in acetate production and increase in propionate
production, which resulted in a decreased acetate to propionate ratio (P < 0:001). TL,
similar to YG, had no effect on in vitro gas production, IVTD and total VFA production
(Table 5). However, K-TL depressed in vitro gas production, IVTD, and total VFA
production.
The negative effects of K-YG (Table 4, Fig. 2) and K-TL (Table 5, Fig. 3) on in vitro gas
production are consistent with observations for K-CO (Table 3). Jenkins (1987) compared
fatty acids differing in their degree of unsaturation using mixtures of oleic and stearic acids,
or linoleic and stearic acids, on in vitro fermentation. As the level of unsaturation increased,

Table 4
Effect of level of YG and potassium soaps of YG in vitro gas production in vitro true digestibility (IVTD) and
volatile fatty acid (VFA) production on incubation of mixed ration in buffered rumen fluid (experiment 3)

Parameters Intercepta Slopeb P-valuec S.E.

YG YG-soap YG YG-soap

Gas production (ml/24 h/g DM) 249.9 0.22 4.6 <0.01 <0.001 4.1
IVTD (DM, per percent) 78.1 0.0 0.24 0.94 <0.001 1.6
VFA (mmol/g DM)
Total 5.4 0.001 0.02 0.87 0.01 0.32
Acetate 3.0 0.005 0.05 0.29 <0.001 0.20
Propionate 1.69 0.0 0.05 0.51 <0.001 0.17
Butyrate 0.50 0.0 0.016 0.17 <0.001 0.03
iso-Valerate 0.09 0.0 0.005 0.58 <0.001 0.02
Valerate 0.10 0.0 0.003 0.88 <0.001 0.02
Acetate:propionate 1.79 0.005 0.05 0.06 <0.001 0.12
Ammonia-N (mg/l) 201.4 0.79 1.6 <0.01 <0.001 11.9
a
The intercept is the fitted value at zero level of fatty acids either from YG or YG-soaps.
b
The slope is the unit of change per percent increase in fatty acids per gram of DM feed.
c
The P-value indicates the probability that the slope differs from zero.
10 G. Getachew et al. / Animal Feed Science and Technology 93 (2001) 115

Table 5
Effect of level of TL and potassium soaps of beef TL in vitro gas production in vitro true digestibility (IVTD)
and volatile fatty acid (VFA) production on incubation of mixed ration in buffered rumen fluid (experiment 3)

Parameters Intercepta Slopeb P-valuec S.E.

TL TL-soap TL TL-soap

Gas production (ml/24 h/g DM) 260.4 0.05 5.3 0.73 <0.001 7.6
IVTD (DM, per percent) 78.6 0.09 0.25 0.10 <0.001 2.28
VFA (mmol/g DM)
Total 5.9 0.009 0.03 0.17 <0.001 0.28
Acetate 3.5 0.001 0.06 0.78 <0.001 0.21
Propionate 1.66 0.009 0.05 <0.01 <0.001 0.13
Butyrate 0.53 0.001 0.013 0.20 <0.001 0.05
iso-Valerate 0.11 0.0 0.004 0.36 <0.001 0.01
Valerate 0.10 0.0 0.002 0.71 <0.001 0.02
Acetate:propionate 2.1 0.009 0.06 <0.01 <0.001 0.12
Ammonia-N (mg/l) 224.8 0.30 1.7 0.44 <0.001 16.3
a
The intercept is the fitted value at zero level of fatty acids either from TL or TL-soaps.
b
The slope is the unit of change per percent increase in fatty acids per gram of DM feed.
c
The P-value indicates the probability that the slope differs from zero.

Fig. 2. The effect of level of fatty acids in YG and YG-soap on in vitro gas production.
 G. Getachew et al. / Animal Feed Science and Technology 93 (2001) 115 11

Fig. 3. The effect of level of fatty acids in TL and TL-soap on in vitro gas production.

concentrations of acetic and butyric acids increased and propionic acid decreased.
However, total VFA production and NDF digestibility were not measured. Earlier, Chalupa
et al. (1984) had observed that oleic acid in the free form decreased the acetate to
propionate ratio, compared to both the control and to stearic acid in the free form, but total
VFA production was unaffected by either free acid of oleic acid or stearic acid. A lack of
change in total VFA production and/or a change in proportion of individual VFAs cannot
support a general conclusion that the effect of fat was detrimental to the fermentation.
Changes in concentrations of individual VFA (Chalupa et al., 1984; Jenkins, 1987) and
changes in production of individual VFA observed in the present study merely indicate a
shift in microbial populations in the rumen in response to fat (Doreau and Chilliard, 1997)
rather than a general suppression in microbial activity.
The depression in ammonia-N (Tables 35) due to addition of fat in the form of K-soaps
is consistent with the ndings of Kowalczyk et al. (1977), and Broudiscou et al. (1990) who
observed decreased ammonia-N production from in vitro fermentation of casein when soy
oil hydrolysate was added. Demeyer and Van Nevel (1995) proposed that this was a
consequence of decreased deaminase activity rather than reduced proteolysis. Neither
calcium soaps nor animalvegetable blend fats inuenced ammonia-N, VFA concentra-
tion, ber digestion or efciency of microbial protein synthesis (Ohajuruka et al., 1991).
Hydrogenation of polyunsaturated fatty acids in the rumen lowers methane production by
competing for hydrogen thereby increasing efciency of utilization of feeds (Blaxter and
Czerkawski, 1966). Since acetate production is associated with generation of relatively
large amounts of hydrogen and CO2, which is used in methanogenesis, reducing acetate
12 G. Getachew et al. / Animal Feed Science and Technology 93 (2001) 115

production by inclusion of fat in the form of soaps should be benecial in terms of

efciency of feed utilization.

4. General discussion

There is a lack of evidence (DePeters and Cant, 1992) to support a general theory that
added fat exerts adverse effects on rumen microbial activity, particularly when supple-
mental fat is included at levels typically fed in commercial dairy rations. Avila et al.
(2000) compared addition of TL or YG to diets of lactating cows and observed no change
in microbial protein production and no effect on ruminal NDF digestibility. Likewise,
infusing canola oil into the rumen did not affect microbial protein synthesis in the rumen
compared to a control diet with no added canola oil (Essex, 1998). Calcium soaps were
used to reduce the biological activity of fatty acids in the rumen (Jenkins and Palmquist,
1984; Alba et al., 1997; Moallem et al., 1997) based on the assumption that divalent
cations react with fatty acids to cause formation of insoluble soaps, which are inert at
normal rumen pH but are converted to free fatty acids and calcium ions in the abomasum
(Jenkins and Palmquist, 1984). However, no difference in DM digestibility between fatty
acids and calcium soaps was observed (Jenkins and Palmquist, 1984) suggesting that fats
in the form of triglycerides do not exert severe negative effects on rumen microbial
activity.
The effect of fat on microbial yield was not measured in the current study, but
published reports from in vivo experiments (Jenkins and Palmquist, 1984; Boggs et al.,
1987; Murphy et al., 1987) indicate that addition of fat increased microbial nitrogen ow
to the lower gut. The low ammonia-N concentrations observed in the current study when
K-soaps were used could be the result of either increased N uptake for microbial growth
or reduce protein degradation. Increased microbial growth resulted when fat was fed
(Ikwuegbu and Sutton, 1982; Machmueller et al., 1998) which suggests that the lower
ammonia-N concentration could be due to increased N uptake rather than reduced protein
degradation.
The absence of negative effects on rumen fermentation parameters with inclusion of fat
in the form of triglycerides reported in this study, and by others (Palmquist and Conrad,
1978; Hussein et al., 1995; Jenkins and Palmquist, 1984; Bateman and Jenkins, 1998),
suggests that triglycerides have less effect on rumen fermentation parameters compared to
the corresponding free fatty acids. This may indicate that the triglycerides were not
completely hydrolyzed to release free fatty acids in the in vitro system or that the fatty acids
were released at a rate sufciently slow for ruminal microbes to hydrogenate them before
they exerted a negative effects. Harfoot (1978) reported in a review that triglycerides are
rapidly hydrolyzed in the rumen, although actual data indicate that the rumen hydrolysis
rates of fats differ (Gerson et al., 1985). Demeyer and Van Nevel (1995) reported that
specicity of lipases for unsaturated fatty acids existed in vitro, thereby suggesting a
preferential release of unsaturated fatty acids. In contrast to the triglyceride form of fats,
the K-soaps dissociated in the rumen buffer thus fatty acids were in their free form and
depressed VFA production as well as gas production (Tables 35). A free carboxyl group
was proposed to be necessary to inhibit microbial growth (Demeyer and Van Nevel, 1995)
 G. Getachew et al. / Animal Feed Science and Technology 93 (2001) 115 13

explaining the negative effect of the K-soaps on in vitro fermentation parameters. The rate
and extent of hydrolysis of triglycerides both in vivo and in vitro warrants further
investigation.

5. Conclusions

Addition of YG and TL fatty acids in the form of triglycerides to in vitro rumen

incubations did not affect in vitro gas production, in vitro true digestibility of DM, and total
VFA production. However, fatty acids of YG and TL in their K-soap form depressed gas
production, IVTD, total VFA production, and ammonia-N concentration. Fatty acids of
CO, a relatively more unsaturated fat than YG and TL, increased in vitro gas production but
had no effect on production of total VFA. K-soaps of CO depressed in vitro gas as well as
VFA production. Although it is often reported and generally believed, that dietary fats have
negative effects on rumen microbial growth and ber digestion, this generalization is not
supported by our results, which only indicate a potential shift in rumen microbial species.
The absence of negative effects on rumen fermentation parameters by inclusion of fat in the
form of triglycerides, suggests that triglycerides have much less effect on rumen fermenta-
tion parameters than their corresponding free fatty acids. Although, triglyceride sources
vary in their effect on the rumen microbial ecosystem, there are currently no methods to
predict the impact of specic fats on rumen fermentation.

Acknowledgements

The authors thank J. Pareas for her help with the rumen stulated animals, D. Daley of
PM AG Products for supplying the fats, and D. Palmquist for his help. Research was
partially supported by the California Dairy Research Foundation and the California
Agricultural Experiment Station. This research was a contribution to W-181 Multistate
Research.

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