Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol85.Issue3-4.Jun2000:
Animal Feed Science and Technology
85 (2000) 153±169
Effects of raw and modi®ed canola lecithins compared
to canola oil, canola seed and soy lecithin on
ruminal fermentation measured with
rumen simulation technique
H.-R. Wettstein, Andrea MachmuÈller, M. Kreuzer*
Institute of Animal Sciences, Animal Nutrition, ETH Zurich, ETH centre/LFW, CH-8092 Zurich, Switzerland
Received 12 October 1999; received in revised form 16 March 2000; accepted 20 April 2000
Abstract
The effects of four different canola lecithins applied at proportions of 30 g fatty acid kgÿ1 diet
were compared with diets containing either no additional lipid or the same amount of fatty acids
from canola seed, pure canola oil and deoiled soy lecithin, respectively. Four types of canola
lecithin with increasing dispersibility in water were used: raw; deoiled; deoiled/hydrolysed; and
hydrolysed/acetylated lecithin. The complete rations consisted of maize silage, hay and concentrate,
and were simultaneously applied in 10 days lasting experimental periods in rumen simulation
technique (Rusitec) with eight consecutive replications each. Like canola seed and pure canola oil,
the lecithins also increased rumen ¯uid pH and propionate proportion of volatile fatty acids (VFA)
whereas total VFA concentration and butyrate proportion were reduced. The level of effect of the
canola lecithins on VFA concentration as well as on bacteria and ciliate count depended on the type
of lecithin. A decrease in ammonia concentration was found with canola oil and all lecithins but not
with canola seed. Compared with the unsupplemented diet, canola oil decreased both acetate to
propionate ratio and methane release. The effects against methane were lower with canola lecithins,
particularly when deoiled. The use of the lecithins did not affect ®bre degradation, whereas apparent
protein degradation was signi®cantly lower than in the other treatments. In spite of its much higher
linoleic acid content, deoiled soy lecithin had quite similar effects as deoiled canola lecithin. Overall,
canola lecithins, particularly in a modi®ed form, could be advantageous in comparison with pure oils in
ruminant nutrition in terms of nutrient degradation. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Lecithin; Canola; Protein degradation; Fibre fermentation; Methane; Ruminants
*
Corresponding author. Tel.: 41-1-632-5972; fax: 41-1-632-1128.
E-mail address: michael.kreuzer@inw.agrl.ethz.ch (M. Kreuzer)
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 4 9 - 8
154
H.-R. Wettstein et al. / Animal Feed Science and Technology 85 (2000) 153±169
1. Introduction
Although plant lipids are high in energy content, in ruminant nutrition their use as pure
oils is restricted by their adverse effects on rumen fermentation, particularly on ®bre
degradation (Jouany, 1994). This could partly result from attachment of lipids to ®bre
(possibly even coating of ®bre) and from a direct antimicrobial effect of fatty acids
(Jenkins, 1993). During processing of raw plant oils, lipid residues are obtained as byproducts. These residues, commonly called lecithins, are complex mixtures mainly of
different phospholipids but also of other polar substances and, if not further processed,
residues of triglycerides (Schol®eld, 1985; Pardun, 1988). Different from oils, lecithins
are dispersible in water (Pardun, 1988) and contain a slowly disappearing fraction
(Jenkins et al., 1989). Therefore, when lecithins are used, attachment to feed particles or
rumen microbes might be less pronounced and the release of the fatty acids could be
delayed resulting in less adverse effects on rumen fermentation (Jenkins, 1993; Nagaraja
et al., 1997). Owing to their af®nity to proteins because of the amphiphatic properties
(Jenkins et al., 1989) and their ability to enhance the formation of a particulate fraction of
protein (Ono et al., 1996), also effects on rumen degradability of protein could occur as
were found by Jenkins and Fotouhi (1990). At present, investigations on the effects of
plant lecithins in rumen fermentation are restricted to a limited number of studies with
soy lecithin (e.g. Yoon et al., 1986; Jenkins et al., 1989; Jenkins and Fotouhi, 1990). Soy
lecithin, however, might have an additional inhibitory effect on the cellulolytic microbes
by its high content of linoleic acid which is much lower in canola lecithin. Technological
modi®cation of plant lecithins furthermore opens the opportunity to alter lecithin
properties towards a better suitability for use in ruminant feeding by reducing their fatty
acid content by means of the transformation of di- to monoglycerides (hydrolysation,
followed by deoiling) and by increasing their dispersibility in water through deoiling,
hydrolysation and acetylation. However, investigations on the effects of lecithin modi®cation are still lacking. The objective of the present study was to compare the effects on
rumen fermentation and nutrient degradation of various canola lecithins with effects of
other lipids using rumen simulation technique (Rusitec; Czerkawski and Breckenridge,
1977). Focus was put on traits which are related to ®bre digestion and protein degradation.
2. Materials and methods
In the present study four types of canola lecithins were employed: raw (CLr); deoiled
(CLd); deoiled/partially hydrolysed (CLd/h); and partially hydrolysed/acetylated canola
lecithin (CLh/a). These lecithins represented an increasing hydrophilic±lipophilic balance
(HLB value, Heusch, 1993) which is a rough indicator of the dispersibility of lecithin
emulsi®ers in water. To be able to separate the effects of the fatty acids from the effects of
the lecithins, a non-supplemented control diet and, as further lipid sources, whole crushed
canola seed (CS), pure canola oil (CO) and deoiled soy lecithin (SLd) were included in
the experiment. Like in the lecithins, the fatty acids present in crushed canola seed are
considered to have weaker effects on rumen fermentation than those of pure canola oil
because of their partially rumen-protected nature (Murphy et al., 1987). All canola
155
H.-R. Wettstein et al. / Animal Feed Science and Technology 85 (2000) 153±169
Table 1
Analytical description of the lipid sources used
Canola
seed (CS)
Canola
oil (CO)
Fatty acid content
0.46
1.00
(relative to CO)
Fatty acids (g kgÿ1 total fatty acids)
C16:0
48
47
C18:0
16
17
C18:1
604
609
C18:2
206
207
C18:3
89
85
Acetone insoluble
n.d.
n.d.a
fraction (g kgÿ1)
a
Canola lecithins
Raw
(CLr)
0.62
77
20
506
296
67
602
Deoiled
(CLd)
0.48
94
13
477
336
53
789
Deoiled/
hydrolysed
(CLd/h)
0.52
107
14
496
303
47
950
Hydrolysed/
acetylated
(CLh/a)
0.62
79
15
517
293
67
603
Soy lecithin
deoiled (SLd)
0.52
181
44
95
577
78
973
Not determined.
lecithins originated from the same batch of raw canola lecithin and were produced like
the soy lecithin by Lucas Meyer GmbH & Co. (Hamburg, Germany).
Table 1 describes the lipid sources used. As expected the fatty acid compositions of
canola seed and oil were very similar whereas the canola lecithins differed in fatty acid
composition from canola oil by higher proportions of saturated fatty acids (Pardun, 1988).
However, the differences to soy lecithin were far higher. Soy lecithin had very low
proportions of oleic acid and relatively high proportions of linoleic acid (0.58) and
palmitic acid (0.18). Modi®cation of the canola lecithins had only small effects on fatty
acid pro®le. Apart from differences in fatty acid content and composition, the lecithins
used differed in the proportion of the acetone insoluble fraction which is a measure of
their content of phospholipids. The lower acetone insoluble fraction of the deoiled canola
lecithin compared with the deoiled soy lecithin was not a result of a higher triglyceride
content due to incomplete deoiling but of the presence of acetone soluble glycolipids in
the deoiled canola lecithin.
All supplemented diets were designed to contain 30 g kgÿ1 fatty acids from canola or
soybean. Accordingly, the complete diets contained between 50 and 61 g kgÿ1 of
lecithins (Table 2) depending on their content of fatty acids in relation to canola oil
(Table 1). The diets consisted of maize silage, hay and the respective concentrate. Apart
from the lipid supplementation the ingredients used for the concentrates were either
wheat and soybean meal (control) or barley, soybean meal and potato protein. All diets
were designed to provide net energy, nitrogen and metabolisable protein in the same ratio
as is recommended for fattening cattle of 300 kg live weight (FAG, 1994). Consequently,
proportion and composition of concentrate were somewhat different in the control diet
from that of the lipid supplemented diets.
A Rusitec system as described by MachmuÈller et al. (1998) was used to monitor the
effects of the dietary treatments on rumen fermentation. The system was equipped with
eight fermenters which allowed the simultaneous evaluation of all eight diets. Eight
156
H.-R. Wettstein et al. / Animal Feed Science and Technology 85 (2000) 153±169
Table 2
Daily quantities and composition of the diets supplied to the individual fermenters
Treatmenta
Control
CS
CO
CLr
CLd
CLd/h
CLh/a
SLd
Quantities
Dry matter (g per day)
Organic matter (g per day)
Calculated net energy (kJ per day)
16.7
16.0
120
16.6
15.7
122
16.6
15.8
121
16.9
16.0
123
17.1
16.2
124
17.1
16.2
124
16.9
16.0
123
17.1
16.2
124
597
107
296
168
±
128
±
±
±
602
154
244
±
30
107
30
77
±
618
154
228
±
52
122
23
±
31
606
151
243
±
51
120
22
±
±
600
149
251
±
50
119
22
±
±
599
149
252
±
50
119
22
±
±
606
151
243
±
51
120
22
±
±
600
149
251
±
50
119
22
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
50
±
±
±
±
±
60
±
±
±
±
±
61
±
±
±
±
±
50
±
±
±
±
±
60
Chemical composition of the concentrates (g kgÿ1 dry matter)
Crude protein
309
340
336
Ether extract
26
192
175
317
177
313
167
315
151
317
176
312
166
Chemical composition of the diets (g kgÿ1 dry matter)
Organic matter
954
949
Gross energy (MJ kgÿ1 DM)
18.8
19.7
Crude protein
146
157
Ether extract
26
61
NDFb
350
371
ADFb
194
214
Cellulose
174
190
Hemicellulose
156
158
Non-NDF carbohydrates
432
360
949
19.6
148
59
368
208
187
160
374
947
19.5
149
58
364
206
185
158
376
945
19.5
149
54
364
206
185
158
378
948
19.6
148
59
368
208
187
160
373
945
19.6
148
58
364
206
185
158
375
Ingredients (g kgÿ1 dry matter)
Maize silage
Hay
Concentrate
Wheat
Barley
Soybean meal
Potato protein
Canola seed
Canola oil
Canola lecithins
Raw
Deoiled
Deoiled/hydrolysed
Hydrolysed/acetylated
Soy lecithin, deoiled
a
b
950
19.6
148
56
376
212
191
163
370
For abbreviations see Table 1.
NDF: neutral detergent ®bre; ADF: acid detergent ®bre.
replicates per treatment were obtained in eight subsequent experimental periods of 10
days each with the ®rst 3 days serving for adaptation of fermentation to the respective
diets. A 3-day adaptation has been found in previous studies (MachmuÈller et al., 1998) to
be the best compromise considering also the limitations to extend the periods beyond 10
days. All fermenters had a capacity of 1.1 l and were ®lled with 800 ml strained rumen
¯uid from one donor cow and 100 ml McDougall buffer (Czerkawski and Breckenridge,
1977) when starting the experimental periods. The buffer ¯ow rate was 510 ml per day,
and the nylon bags (70140 mm) had a pore size of 100 mm as recommended by Carro
et al. (1995). At the beginning of each experimental period one of the two nylon bags was
H.-R. Wettstein et al. / Animal Feed Science and Technology 85 (2000) 153±169
157
®lled with 80 g solid rumen content and the other one with the respective diet. Every day
the respective ®rst introduced nylon bag was replaced starting with the one ®lled with
rumen content thus achieving a general feed incubation period of 48 h. The feed
ingredients were mixed before starting the experimental period and daily portions were
frozen until application. Prior to mixing with the other ingredients for each experimental
period the whole batches of maize silage and hay were structurally fractured to 0.3 and
0.5 cm particle length, respectively, in a food mixer equipped with a cutting blade
(Moulinette1S, Group Moulinex, Paris, France). The diets were supplied at daily
amounts of ca. 17 g dry matter depending on their energy content (Table 2).
Rumen ¯uid was sampled with a syringe and a infusion tube which was inserted
through a three-way tap into the fermenter 2 h prior to the daily exchange of feed bags. In
rumen ¯uid pH, ammonia and redox potential (to control anaerobic conditions) were
measured with a pH meter (model 713, Metrom, Herisau, Switzerland) equipped with the
respective electrodes. For determination of volatile fatty acids, 1.8 ml samples of rumen
¯uid were stabilised with 200 ml 46 mM HgCl2-solution and frozen until analysis with
gas chromatography (GC Star 3400 CX, Varian, Palo Alto, CA, USA) according to
Tangerman and Nagengast (1996) after formic acid acidi®cation of the samples. Bacteria
and protozoa were counted using a BuÈrker counting chamber (depth 0.1 mm, Blau Brand,
Wertheim, Germany). Ciliates were classi®ed into holotrichs and entodiniomorphs. The
total gas produced in each fermenter was collected in gas proof bags (TECOBAG 5 L,
PETP/AL/PE Ð 12/12/75 quality, Tesseraux Container GmbH, BuÈrstadt, Germany). Gas
production was quanti®ed by the respective replacement of water. In the gas samples,
contents of methane and hydrogen were analysed by gas chromatography (model 5890
Series II, Hewlett-Packard, Wilmington, DE, USA) equipped with a molecular sieve 13 X
column, a FID and a WLD detector. With the exception of bacteria counts (determined on
Day 0, 5 and 9 only) daily measurements of all parameters were carried out for each
fermenter.
Diets and fermentation residues were lyophilised and analysed for dry matter, ash, total
lipids (Soxhlet method) and crude protein (Kjeldahl method) according to standard
techniques (Naumann and Bassler, 1997). Furthermore, contents of gross energy
(anisotherm bomb calorimetry, IKA C 700 T, IKA-Werke GmbH & Co. KG, Staufen,
Germany), ash free NDF, ADF and ADL (Robertson and van Soest, 1981) were
determined after incubation with amylase. From the detergent fractions, hemicellulose
(NDF±ADF) and cellulose (ADF±ADL) were calculated. In the lipid supplements,
content and composition of fatty acids were analysed with gas chromatography (model
5890, Hewlett-Packard, Wilmington, DE, USA) after methylating the fatty acids using
boron tri¯uoride as described by Gebert et al. (1999). C13:0 methyl ester was used as
internal standard. For the determination of the acetone insoluble fraction of the lecithins
by the recommended method (Lucas Meyer, Hamburg, Germany), 20 ml acetone of 08C
were mixed with 5 g of lecithin. After sedimentation the supernatant was ®ltered (589/2
white ribbon ®lter, Schleicher & SchuÈll, Dassel, Germany). This procedure was repeated
for at least ®ve times. When the insoluble part disintegrated into a ®ne powder it was
brought to the ®lter too and washed with cold acetone until the ®ltered acetone
evaporated without any residues. Residues on the ®lter representing the acetone insoluble
fraction were dried for 30 min at 1058C under vacuum and weighed after cooling.
158
H.-R. Wettstein et al. / Animal Feed Science and Technology 85 (2000) 153±169
The apparent degradation of nutrients in the nylon bags were determined from the bag
residues pooled from Days 4 to 10. Hydrogen balance was calculated according to
Demeyer (1991) considering the main volatile fatty acids (acetate, propionate and
butyrate) and methane production. Further details on the experimental and analytical
techniques are given elsewhere (MachmuÈller et al., 1998).
Statistical evaluation was carried out by analysis of variance regarding diet and
experimental period as effects. The GLM-procedure of SAS (version 6.10, SAS Institute
Inc., Cary, NC, USA) was applied. The treatment means were statistically compared with the
Tukey method. The tables give the group means and the standard errors of mean (SEM).
3. Results
3.1. Rumen ¯uid properties
Generally, the redox potential of rumen ¯uid was clearly negative accounting for
ÿ21427 mV on average (data not shown in table). Rumen ¯uid pH was signi®cantly
lower with the unsupplemented control diet than with raw canola lecithin (Table 3). For
all other lipid supplemented diets rumen ¯uid pH also ranged at a relatively high level,
but this was not signi®cantly different from control. With all lecithins and with canola oil
(CO) ammonia concentration in rumen ¯uid was signi®cantly reduced by 2.0±
2.9 mmol lÿ1 when compared with control and canola seed. In VFA production per
day the difference to the control was signi®cant for all lipid supplemented diets except for
the hydrolysed/acetylated canola lecithin. In the rumen ¯uid receiving the control diet,
the concentration of volatile fatty acids (VFA) was signi®cantly higher than with canola
oil and raw canola lecithin. For the other lecithin supplemented diets and the canola seed
diet the values of VFA concentration were intermediate. The proportion of acetate was
somewhat higher in the supplemented diets than in control, but without reaching the level
of signi®cance. Compared with control, all supplemented diets had signi®cantly higher
proportions of propionate (2.2 to 3.1%) and signi®cantly lower proportions of
butyrate (ÿ3.2 to ÿ4.2%). In the canola seed treatment the highest proportion of iso-acids
occurred. While the difference between canola seed and canola oil as well as all lecithins
was signi®cant for iso-butyrate, it was signi®cant between canola seed and control as well
as all lecithins for iso-valerate. The acetate to propionate ratio was lower in all lipid
supplemented diets than with the unsupplemented control. Bacteria counts were reduced
with pure canola oil and canola seed as well as with all lecithin containing diets. The
effect was most pronounced with raw and deoiled (CLd) canola lecithin. There was a
signi®cant treatment effect (p
85 (2000) 153±169
Effects of raw and modi®ed canola lecithins compared
to canola oil, canola seed and soy lecithin on
ruminal fermentation measured with
rumen simulation technique
H.-R. Wettstein, Andrea MachmuÈller, M. Kreuzer*
Institute of Animal Sciences, Animal Nutrition, ETH Zurich, ETH centre/LFW, CH-8092 Zurich, Switzerland
Received 12 October 1999; received in revised form 16 March 2000; accepted 20 April 2000
Abstract
The effects of four different canola lecithins applied at proportions of 30 g fatty acid kgÿ1 diet
were compared with diets containing either no additional lipid or the same amount of fatty acids
from canola seed, pure canola oil and deoiled soy lecithin, respectively. Four types of canola
lecithin with increasing dispersibility in water were used: raw; deoiled; deoiled/hydrolysed; and
hydrolysed/acetylated lecithin. The complete rations consisted of maize silage, hay and concentrate,
and were simultaneously applied in 10 days lasting experimental periods in rumen simulation
technique (Rusitec) with eight consecutive replications each. Like canola seed and pure canola oil,
the lecithins also increased rumen ¯uid pH and propionate proportion of volatile fatty acids (VFA)
whereas total VFA concentration and butyrate proportion were reduced. The level of effect of the
canola lecithins on VFA concentration as well as on bacteria and ciliate count depended on the type
of lecithin. A decrease in ammonia concentration was found with canola oil and all lecithins but not
with canola seed. Compared with the unsupplemented diet, canola oil decreased both acetate to
propionate ratio and methane release. The effects against methane were lower with canola lecithins,
particularly when deoiled. The use of the lecithins did not affect ®bre degradation, whereas apparent
protein degradation was signi®cantly lower than in the other treatments. In spite of its much higher
linoleic acid content, deoiled soy lecithin had quite similar effects as deoiled canola lecithin. Overall,
canola lecithins, particularly in a modi®ed form, could be advantageous in comparison with pure oils in
ruminant nutrition in terms of nutrient degradation. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Lecithin; Canola; Protein degradation; Fibre fermentation; Methane; Ruminants
*
Corresponding author. Tel.: 41-1-632-5972; fax: 41-1-632-1128.
E-mail address: michael.kreuzer@inw.agrl.ethz.ch (M. Kreuzer)
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 4 9 - 8
154
H.-R. Wettstein et al. / Animal Feed Science and Technology 85 (2000) 153±169
1. Introduction
Although plant lipids are high in energy content, in ruminant nutrition their use as pure
oils is restricted by their adverse effects on rumen fermentation, particularly on ®bre
degradation (Jouany, 1994). This could partly result from attachment of lipids to ®bre
(possibly even coating of ®bre) and from a direct antimicrobial effect of fatty acids
(Jenkins, 1993). During processing of raw plant oils, lipid residues are obtained as byproducts. These residues, commonly called lecithins, are complex mixtures mainly of
different phospholipids but also of other polar substances and, if not further processed,
residues of triglycerides (Schol®eld, 1985; Pardun, 1988). Different from oils, lecithins
are dispersible in water (Pardun, 1988) and contain a slowly disappearing fraction
(Jenkins et al., 1989). Therefore, when lecithins are used, attachment to feed particles or
rumen microbes might be less pronounced and the release of the fatty acids could be
delayed resulting in less adverse effects on rumen fermentation (Jenkins, 1993; Nagaraja
et al., 1997). Owing to their af®nity to proteins because of the amphiphatic properties
(Jenkins et al., 1989) and their ability to enhance the formation of a particulate fraction of
protein (Ono et al., 1996), also effects on rumen degradability of protein could occur as
were found by Jenkins and Fotouhi (1990). At present, investigations on the effects of
plant lecithins in rumen fermentation are restricted to a limited number of studies with
soy lecithin (e.g. Yoon et al., 1986; Jenkins et al., 1989; Jenkins and Fotouhi, 1990). Soy
lecithin, however, might have an additional inhibitory effect on the cellulolytic microbes
by its high content of linoleic acid which is much lower in canola lecithin. Technological
modi®cation of plant lecithins furthermore opens the opportunity to alter lecithin
properties towards a better suitability for use in ruminant feeding by reducing their fatty
acid content by means of the transformation of di- to monoglycerides (hydrolysation,
followed by deoiling) and by increasing their dispersibility in water through deoiling,
hydrolysation and acetylation. However, investigations on the effects of lecithin modi®cation are still lacking. The objective of the present study was to compare the effects on
rumen fermentation and nutrient degradation of various canola lecithins with effects of
other lipids using rumen simulation technique (Rusitec; Czerkawski and Breckenridge,
1977). Focus was put on traits which are related to ®bre digestion and protein degradation.
2. Materials and methods
In the present study four types of canola lecithins were employed: raw (CLr); deoiled
(CLd); deoiled/partially hydrolysed (CLd/h); and partially hydrolysed/acetylated canola
lecithin (CLh/a). These lecithins represented an increasing hydrophilic±lipophilic balance
(HLB value, Heusch, 1993) which is a rough indicator of the dispersibility of lecithin
emulsi®ers in water. To be able to separate the effects of the fatty acids from the effects of
the lecithins, a non-supplemented control diet and, as further lipid sources, whole crushed
canola seed (CS), pure canola oil (CO) and deoiled soy lecithin (SLd) were included in
the experiment. Like in the lecithins, the fatty acids present in crushed canola seed are
considered to have weaker effects on rumen fermentation than those of pure canola oil
because of their partially rumen-protected nature (Murphy et al., 1987). All canola
155
H.-R. Wettstein et al. / Animal Feed Science and Technology 85 (2000) 153±169
Table 1
Analytical description of the lipid sources used
Canola
seed (CS)
Canola
oil (CO)
Fatty acid content
0.46
1.00
(relative to CO)
Fatty acids (g kgÿ1 total fatty acids)
C16:0
48
47
C18:0
16
17
C18:1
604
609
C18:2
206
207
C18:3
89
85
Acetone insoluble
n.d.
n.d.a
fraction (g kgÿ1)
a
Canola lecithins
Raw
(CLr)
0.62
77
20
506
296
67
602
Deoiled
(CLd)
0.48
94
13
477
336
53
789
Deoiled/
hydrolysed
(CLd/h)
0.52
107
14
496
303
47
950
Hydrolysed/
acetylated
(CLh/a)
0.62
79
15
517
293
67
603
Soy lecithin
deoiled (SLd)
0.52
181
44
95
577
78
973
Not determined.
lecithins originated from the same batch of raw canola lecithin and were produced like
the soy lecithin by Lucas Meyer GmbH & Co. (Hamburg, Germany).
Table 1 describes the lipid sources used. As expected the fatty acid compositions of
canola seed and oil were very similar whereas the canola lecithins differed in fatty acid
composition from canola oil by higher proportions of saturated fatty acids (Pardun, 1988).
However, the differences to soy lecithin were far higher. Soy lecithin had very low
proportions of oleic acid and relatively high proportions of linoleic acid (0.58) and
palmitic acid (0.18). Modi®cation of the canola lecithins had only small effects on fatty
acid pro®le. Apart from differences in fatty acid content and composition, the lecithins
used differed in the proportion of the acetone insoluble fraction which is a measure of
their content of phospholipids. The lower acetone insoluble fraction of the deoiled canola
lecithin compared with the deoiled soy lecithin was not a result of a higher triglyceride
content due to incomplete deoiling but of the presence of acetone soluble glycolipids in
the deoiled canola lecithin.
All supplemented diets were designed to contain 30 g kgÿ1 fatty acids from canola or
soybean. Accordingly, the complete diets contained between 50 and 61 g kgÿ1 of
lecithins (Table 2) depending on their content of fatty acids in relation to canola oil
(Table 1). The diets consisted of maize silage, hay and the respective concentrate. Apart
from the lipid supplementation the ingredients used for the concentrates were either
wheat and soybean meal (control) or barley, soybean meal and potato protein. All diets
were designed to provide net energy, nitrogen and metabolisable protein in the same ratio
as is recommended for fattening cattle of 300 kg live weight (FAG, 1994). Consequently,
proportion and composition of concentrate were somewhat different in the control diet
from that of the lipid supplemented diets.
A Rusitec system as described by MachmuÈller et al. (1998) was used to monitor the
effects of the dietary treatments on rumen fermentation. The system was equipped with
eight fermenters which allowed the simultaneous evaluation of all eight diets. Eight
156
H.-R. Wettstein et al. / Animal Feed Science and Technology 85 (2000) 153±169
Table 2
Daily quantities and composition of the diets supplied to the individual fermenters
Treatmenta
Control
CS
CO
CLr
CLd
CLd/h
CLh/a
SLd
Quantities
Dry matter (g per day)
Organic matter (g per day)
Calculated net energy (kJ per day)
16.7
16.0
120
16.6
15.7
122
16.6
15.8
121
16.9
16.0
123
17.1
16.2
124
17.1
16.2
124
16.9
16.0
123
17.1
16.2
124
597
107
296
168
±
128
±
±
±
602
154
244
±
30
107
30
77
±
618
154
228
±
52
122
23
±
31
606
151
243
±
51
120
22
±
±
600
149
251
±
50
119
22
±
±
599
149
252
±
50
119
22
±
±
606
151
243
±
51
120
22
±
±
600
149
251
±
50
119
22
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
50
±
±
±
±
±
60
±
±
±
±
±
61
±
±
±
±
±
50
±
±
±
±
±
60
Chemical composition of the concentrates (g kgÿ1 dry matter)
Crude protein
309
340
336
Ether extract
26
192
175
317
177
313
167
315
151
317
176
312
166
Chemical composition of the diets (g kgÿ1 dry matter)
Organic matter
954
949
Gross energy (MJ kgÿ1 DM)
18.8
19.7
Crude protein
146
157
Ether extract
26
61
NDFb
350
371
ADFb
194
214
Cellulose
174
190
Hemicellulose
156
158
Non-NDF carbohydrates
432
360
949
19.6
148
59
368
208
187
160
374
947
19.5
149
58
364
206
185
158
376
945
19.5
149
54
364
206
185
158
378
948
19.6
148
59
368
208
187
160
373
945
19.6
148
58
364
206
185
158
375
Ingredients (g kgÿ1 dry matter)
Maize silage
Hay
Concentrate
Wheat
Barley
Soybean meal
Potato protein
Canola seed
Canola oil
Canola lecithins
Raw
Deoiled
Deoiled/hydrolysed
Hydrolysed/acetylated
Soy lecithin, deoiled
a
b
950
19.6
148
56
376
212
191
163
370
For abbreviations see Table 1.
NDF: neutral detergent ®bre; ADF: acid detergent ®bre.
replicates per treatment were obtained in eight subsequent experimental periods of 10
days each with the ®rst 3 days serving for adaptation of fermentation to the respective
diets. A 3-day adaptation has been found in previous studies (MachmuÈller et al., 1998) to
be the best compromise considering also the limitations to extend the periods beyond 10
days. All fermenters had a capacity of 1.1 l and were ®lled with 800 ml strained rumen
¯uid from one donor cow and 100 ml McDougall buffer (Czerkawski and Breckenridge,
1977) when starting the experimental periods. The buffer ¯ow rate was 510 ml per day,
and the nylon bags (70140 mm) had a pore size of 100 mm as recommended by Carro
et al. (1995). At the beginning of each experimental period one of the two nylon bags was
H.-R. Wettstein et al. / Animal Feed Science and Technology 85 (2000) 153±169
157
®lled with 80 g solid rumen content and the other one with the respective diet. Every day
the respective ®rst introduced nylon bag was replaced starting with the one ®lled with
rumen content thus achieving a general feed incubation period of 48 h. The feed
ingredients were mixed before starting the experimental period and daily portions were
frozen until application. Prior to mixing with the other ingredients for each experimental
period the whole batches of maize silage and hay were structurally fractured to 0.3 and
0.5 cm particle length, respectively, in a food mixer equipped with a cutting blade
(Moulinette1S, Group Moulinex, Paris, France). The diets were supplied at daily
amounts of ca. 17 g dry matter depending on their energy content (Table 2).
Rumen ¯uid was sampled with a syringe and a infusion tube which was inserted
through a three-way tap into the fermenter 2 h prior to the daily exchange of feed bags. In
rumen ¯uid pH, ammonia and redox potential (to control anaerobic conditions) were
measured with a pH meter (model 713, Metrom, Herisau, Switzerland) equipped with the
respective electrodes. For determination of volatile fatty acids, 1.8 ml samples of rumen
¯uid were stabilised with 200 ml 46 mM HgCl2-solution and frozen until analysis with
gas chromatography (GC Star 3400 CX, Varian, Palo Alto, CA, USA) according to
Tangerman and Nagengast (1996) after formic acid acidi®cation of the samples. Bacteria
and protozoa were counted using a BuÈrker counting chamber (depth 0.1 mm, Blau Brand,
Wertheim, Germany). Ciliates were classi®ed into holotrichs and entodiniomorphs. The
total gas produced in each fermenter was collected in gas proof bags (TECOBAG 5 L,
PETP/AL/PE Ð 12/12/75 quality, Tesseraux Container GmbH, BuÈrstadt, Germany). Gas
production was quanti®ed by the respective replacement of water. In the gas samples,
contents of methane and hydrogen were analysed by gas chromatography (model 5890
Series II, Hewlett-Packard, Wilmington, DE, USA) equipped with a molecular sieve 13 X
column, a FID and a WLD detector. With the exception of bacteria counts (determined on
Day 0, 5 and 9 only) daily measurements of all parameters were carried out for each
fermenter.
Diets and fermentation residues were lyophilised and analysed for dry matter, ash, total
lipids (Soxhlet method) and crude protein (Kjeldahl method) according to standard
techniques (Naumann and Bassler, 1997). Furthermore, contents of gross energy
(anisotherm bomb calorimetry, IKA C 700 T, IKA-Werke GmbH & Co. KG, Staufen,
Germany), ash free NDF, ADF and ADL (Robertson and van Soest, 1981) were
determined after incubation with amylase. From the detergent fractions, hemicellulose
(NDF±ADF) and cellulose (ADF±ADL) were calculated. In the lipid supplements,
content and composition of fatty acids were analysed with gas chromatography (model
5890, Hewlett-Packard, Wilmington, DE, USA) after methylating the fatty acids using
boron tri¯uoride as described by Gebert et al. (1999). C13:0 methyl ester was used as
internal standard. For the determination of the acetone insoluble fraction of the lecithins
by the recommended method (Lucas Meyer, Hamburg, Germany), 20 ml acetone of 08C
were mixed with 5 g of lecithin. After sedimentation the supernatant was ®ltered (589/2
white ribbon ®lter, Schleicher & SchuÈll, Dassel, Germany). This procedure was repeated
for at least ®ve times. When the insoluble part disintegrated into a ®ne powder it was
brought to the ®lter too and washed with cold acetone until the ®ltered acetone
evaporated without any residues. Residues on the ®lter representing the acetone insoluble
fraction were dried for 30 min at 1058C under vacuum and weighed after cooling.
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H.-R. Wettstein et al. / Animal Feed Science and Technology 85 (2000) 153±169
The apparent degradation of nutrients in the nylon bags were determined from the bag
residues pooled from Days 4 to 10. Hydrogen balance was calculated according to
Demeyer (1991) considering the main volatile fatty acids (acetate, propionate and
butyrate) and methane production. Further details on the experimental and analytical
techniques are given elsewhere (MachmuÈller et al., 1998).
Statistical evaluation was carried out by analysis of variance regarding diet and
experimental period as effects. The GLM-procedure of SAS (version 6.10, SAS Institute
Inc., Cary, NC, USA) was applied. The treatment means were statistically compared with the
Tukey method. The tables give the group means and the standard errors of mean (SEM).
3. Results
3.1. Rumen ¯uid properties
Generally, the redox potential of rumen ¯uid was clearly negative accounting for
ÿ21427 mV on average (data not shown in table). Rumen ¯uid pH was signi®cantly
lower with the unsupplemented control diet than with raw canola lecithin (Table 3). For
all other lipid supplemented diets rumen ¯uid pH also ranged at a relatively high level,
but this was not signi®cantly different from control. With all lecithins and with canola oil
(CO) ammonia concentration in rumen ¯uid was signi®cantly reduced by 2.0±
2.9 mmol lÿ1 when compared with control and canola seed. In VFA production per
day the difference to the control was signi®cant for all lipid supplemented diets except for
the hydrolysed/acetylated canola lecithin. In the rumen ¯uid receiving the control diet,
the concentration of volatile fatty acids (VFA) was signi®cantly higher than with canola
oil and raw canola lecithin. For the other lecithin supplemented diets and the canola seed
diet the values of VFA concentration were intermediate. The proportion of acetate was
somewhat higher in the supplemented diets than in control, but without reaching the level
of signi®cance. Compared with control, all supplemented diets had signi®cantly higher
proportions of propionate (2.2 to 3.1%) and signi®cantly lower proportions of
butyrate (ÿ3.2 to ÿ4.2%). In the canola seed treatment the highest proportion of iso-acids
occurred. While the difference between canola seed and canola oil as well as all lecithins
was signi®cant for iso-butyrate, it was signi®cant between canola seed and control as well
as all lecithins for iso-valerate. The acetate to propionate ratio was lower in all lipid
supplemented diets than with the unsupplemented control. Bacteria counts were reduced
with pure canola oil and canola seed as well as with all lecithin containing diets. The
effect was most pronounced with raw and deoiled (CLd) canola lecithin. There was a
signi®cant treatment effect (p