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 kgÿ1diet 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 Animal Feed Science and Technology
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*Corresponding author. Tel.:41-1-632-5972; fax:41-1-632-1128. E-mail address: michael.kreuzer@inw.agrl.ethz.ch (M. Kreuzer)
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 by-products. 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
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 Table 1
Analytical description of the lipid sources used
Canola
0.46 1.00 0.62 0.48 0.52 0.62 0.52
Fatty acids (g kgÿ1total fatty acids)
C16:0 48 47 77 94 107 79 181
C18:0 16 17 20 13 14 15 44
C18:1 604 609 506 477 496 517 95
C18:2 206 207 296 336 303 293 577
C18:3 89 85 67 53 47 67 78
Acetone insoluble fraction (g kgÿ1)
n.d.a n.d. 602 789 950 603 973
aNot determined.
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 100mm as recommended by Carro et al. (1995). At the beginning of each experimental period one of the two nylon bags was 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) 16.7 16.6 16.6 16.9 17.1 17.1 16.9 17.1 Organic matter (g per day) 16.0 15.7 15.8 16.0 16.2 16.2 16.0 16.2 Calculated net energy (kJ per day) 120 122 121 123 124 124 123 124
Ingredients (g kgÿ1dry matter)
Maize silage 597 602 618 606 600 599 606 600
Hay 107 154 154 151 149 149 151 149
Concentrate 296 244 228 243 251 252 243 251
Wheat 168 ± ± ± ± ± ± ±
Barley ± 30 52 51 50 50 51 50
Soybean meal 128 107 122 120 119 119 120 119
Potato protein ± 30 23 22 22 22 22 22
Canola seed ± 77 ± ± ± ± ± ±
Canola oil ± ± 31 ± ± ± ± ±
Canola lecithins
Raw ± ± ± 50 ± ± ± ±
Deoiled ± ± ± ± 60 ± ± ±
Deoiled/hydrolysed ± ± ± ± ± 61 ± ±
Hydrolysed/acetylated ± ± ± ± ± ± 50 ±
Soy lecithin, deoiled ± ± ± ± ± ± ± 60
Chemical composition of the concentrates (g kgÿ1dry matter)
Crude protein 309 340 336 317 313 315 317 312
Ether extract 26 192 175 177 167 151 176 166
Chemical composition of the diets (g kgÿ1dry matter)
Organic matter 954 949 950 949 947 945 948 945
Gross energy (MJ kgÿ1DM) 18.8 19.7 19.6 19.6 19.5 19.5 19.6 19.6
Crude protein 146 157 148 148 149 149 148 148
Ether extract 26 61 56 59 58 54 59 58
NDFb 350 371 376 368 364 364 368 364
ADFb 194 214 212 208 206 206 208 206
Cellulose 174 190 191 187 185 185 187 185
Hemicellulose 156 158 163 160 158 158 160 158
Non-NDF carbohydrates 432 360 370 374 376 378 373 375
aFor abbreviations see Table 1.
bNDF: neutral detergent ®bre; ADF: acid detergent ®bre.
®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 to0.3 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 200ml 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.
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 toÿ4.2%). 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<0.05) for the number of total ciliates, but no signi®cant differences were identi®ed between individual groups by multiple comparison among means. Both holotrichs and entodiniomorphs tended to be higher in the lecithin groups.
3.2. Gaseous emissions and hydrogen balance
Table 3
Treatment effects on rumen ¯uid characteristics (averages of Days 4±10)a
Treatmentb Control CS CO CL
r CLd CLd/h CLh/a SLd SEM
Rumen ¯uid properties
pH 6.56 b 6.66 ab 6.66 ab 6.66 a 6.65 ab 6.62 ab 6.66 ab 6.65 ab 0.021
Ammonia (mmol lÿ1) 11.3 a 11.6 a 9.3 b 8.8 b 9.2 b 9.3 b 8.4 b 9.2 b 0.23
Volatile fatty acids
Total (mmol per day) 84.0 a 79.7 b 79.4 b 78.0 b 78.8 b 78.8 b 80.5 ab 79.5 b 0.89
Total (mmol lÿ1) 154 a 148 ab 146 b 147 b 148 ab 150 ab 147 ab 148 ab 1.7
Molar proportions (%)
Acetate 49.8 50.5 50.5 51.1 51.7 51.0 50.4 50.7 0.45
Propionate 17.9 b 20.9 a 20.9 a 21.0 a 20.1 a 20.3 a 20.7 a 20.5 a 0.40
n-Butyrate 25.1 a 21.1 b 21.4 b 20.9 b 21.5 b 21.9 b 21.8 b 21.9 b 0.46
iso-Butyrate 0.34 ab 0.35 a 0.31 bc 0.30 c 0.31 bc 0.30 c 0.29 c 0.31 bc 0.006
n-Valerate 5.8 ab 6.0 a 5.9 ab 5.7 ab 5.3 b 5.6 ab 5.7 ab 5.7 ab 0.14
iso-Valerate 1.03 bc 1.17 a 1.09 ab 1.06 bc 1.03 bc 1.00 bc 0.99 bc 0.98 c 0.023
Acetate:propionate, x:1 2.91 a 2.52 b 2.50 b 2.53 b 2.64 b 2.59 b 2.50 b 2.57 b 0.057
Microbial counts
Bacteria (109mlÿ1) 4.04 a 3.86 ab 3.64 abc 3.33 c 3.49 bc 3.66 abc 3.63 abc 3.66 abc 0.111
Ciliates (103mlÿ1)
Holotrichs 0.21 0.41 0.51 0.48 0.75 0.72 0.68 0.44 0.142
Entodiniomorphs 4.06 3.62 3.48 3.85 5.08 4.50 4.54 4.37 0.418
Total 4.26 4.02 3.99 4.33 5.83 5.22 5.22 4.81 0.442
aSEM: standard error of mean. Means by treatment,n8. Mean values within the same line sharing no common letters are signi®cantly different (p<0.05). bFor abbreviations see Table 1.
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Table 4
Treatment effects on fermentation gases (averages of Days 4±10)a
Treatmentb Control CS CO CL
r CLd CLd/h CLh/a SLd SEM
Gas release from rumen ¯uid
Gas production (l per day) 2.26 a 1.96 b 1.99 b 1.97 b 2.03 b 2.01 b 2.04 b 2.02 b 0.038 Methane
Methane (mmol per day) 10.77 a 7.96 c 8.03 c 8.89 bc 9.70 ab 9.32 b 8.64 bc 9.29 b 0.273 aFOM (mmol gÿ1)c 1.19 a 0.95 c 0.93 c 1.07 abc 1.16 ab 1.12 ab 1.03 bc 1.11 ab 0.034
aNDF (mmol gÿ1)d 11.86 a 7.43 b 7.27 b 9.12 b 9.00 b 9.14 b 8.74 b 8.91 b 0.585
Hydrogen
Hydrogen (mmol per day) 0.80 ab 0.92 ab 1.03 ab 0.84 ab 0.64 b 0.79 ab 1.13 a 0.74 ab 0.107 aFOM (mmol gÿ1) 0.09 ab 0.11 ab 0.12 ab 0.10 ab 0.07 b 0.09 ab 0.13 a 0.09 ab 0.012
aNDF (mmol gÿ1) 0.75 ab 0.78 ab 0.82 ab 0.75 ab 0.53 b 0.73 ab 0.95 a 0.65 ab 0.092
Hydrogen balance
Produced (mol per day) 0.200 a 0.180 b 0.180 b 0.176 b 0.179 b 0.184 b 0.180 b 0.181 b 0.0025 Utilised (mol per day) 0.123 a 0.106 b 0.106 b 0.108 b 0.111 b 0.112 b 0.109 b 0.111 b 0.0019
Recovered 0.65 ab 0.64 b 0.64 b 0.68 ab 0.68 ab 0.63 b 0.67 ab 0.71 a 0.016
aSEM: standard error of mean. Means by treatment,n8. Mean values within the same line sharing no common letters are signi®cantly different (p<0.05). bFor abbreviations see Table 1.
caFOM: apparently fermented organic matter. daNDF: apparently fermented neutral detergent ®bre.
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and seed (ÿ30%) followed by hydrolysed/acetylated canola lecithin (ÿ24%) and raw canola lecithin (ÿ21%). Methane suppression was lower with the deoiled lecithins (ÿ13 to ÿ16%). In relation to apparently fermented organic matter, the greatest decline in methane release also occurred with canola oil and canola seed (ÿ21%) followed by hydrolysed/acetylated canola lecithin (ÿ13%). For all other lecithin supplemented diets the decrease was not signi®cant against control. When related to apparently fermented NDF, in all lipid supplemented diets a decline of methane release occurred, which was again highest in the canola oil (ÿ39%) and the canola seed diet (ÿ37%). Hydrogen release from fermentation was lowest with the deoiled canola lecithin (CLd) and highest
with partially hydrolysed/acetylated canola lecithin for production per day and per unit of apparently fermented organic matter as well as NDF. For all other treatments the values were intermediate. In the unsupplemented control diet both hydrogen produced and utilised were higher than in all supplemented treatments. Hydrogen recovery was highest in the soy lecithin treatment and lowest with deoiled partially hydrolysed canola lecithin, canola oil and canola seed.
3.3. Apparent degradation of nutrients
The apparent ruminal degradation of organic matter, as estimated by the percentage disappeared from the nylon bags during 48 h of incubation in the rumen ¯uid, was signi®cantly reduced by each type of lecithin supplementation at an average of 4.5 percentage units (Table 5). The reductions found with canola oil and canola seed were lower accounting for 1.6 and 2.9 percentage units, respectively. Disappearance of gross energy was similarly affected. Apparent degradation of crude protein was signi®cantly lower with all lecithin treatments, averaging at 0.50, compared with the unsupplemented control (0.59) and the canola seed treatment (0.57). Concerning the canola oil treatment only the differences to the canola lecithin diets were signi®cant but not to the soy lecithin group. Ether extract disappearance was highest with the canola oil treatment followed by the unsupplemented control and the not deoiled canola lecithins (CLrand CLh/a). In turn,
ether extract disappearance rate was signi®cantly lowest with all deoiled lecithins. No signi®cant effects on the degradation of different cell wall carbohydrate fractions were found, although the lipid supplemented diets tended to result in a slightly higher ®bre degradation rate. Apparent degradation ranged from 0.19 to 0.20 for hemicellulose and from 0.16 to 0.19 for cellulose. Disappearance of non-NDF carbohydrates was generally high and not signi®cantly different between groups.
4. Discussion
4.1. General effects of canola lipids on rumen fermentation
Table 5
Treatment effects on apparent ruminal nutrient degradation (averages of Days 4±10)a
Treatmentb Control CS CO CLr CLd CLd/h CLh/a SLd SEM
Disappearance rate
Organic matter 0.564 a 0.535 abc 0.548 ab 0.521 bc 0.515 c 0.514 c 0.526 bc 0.517 b 0.0070 Gross energy 0.541 a 0.511 abc 0.529 ab 0.491 cd 0.477 d 0.479 cd 0.499 bcd 0.481 cd 0.0074 Crude protein 0.586 a 0.566 a 0.559 ab 0.506 c 0.496 c 0.494 c 0.491 c 0.516 bc 0.0098 Ether extract 0.429 b 0.453 b 0.554 a 0.391 b 0.287 c 0.306 c 0.426 b 0.305 c 0.0140
NDF 0.159 0.180 0.183 0.170 0.178 0.161 0.171 0.175 0.0083
ADF 0.138 0.157 0.168 0.144 0.156 0.149 0.156 0.150 0.0083
Cellulose 0.160 0.183 0.194 0.173 0.185 0.175 0.181 0.177 0.0087
Hemicellulose 0.186 0.210 0.201 0.202 0.207 0.196 0.190 0.207 0.0099
Non-NDF CHOc 0.892 0.903 0.912 0.894 0.884 0.884 0.906 0.884 0.0084
aSEM: standard error of mean. Means by treatment,n8. Mean values within the same line sharing no common superscript are signi®cantly different (p<0.05). bFor abbreviations see Table 1.
cNon-NDF CHO: non-NDF carbohydrate.
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therefore higher percentage of propionate. While most researchers found a reduction in acetate proportion (Jenkins, 1990; Doreau et al., 1993), in the present experiment the canola oil supplementation only slightly reduced the acetate production and its percentage was not affected. In turn, the canola oil treatment signi®cantly reduced n -butyrate proportion like canola lipids used in previous investigations (Dong et al., 1997; MachmuÈller et al., 1998). In control, then-butyrate concentrations ranged at a similarly high level as found in other Rusitec experiments with maize silage-based diets (Carro et al., 1995; MachmuÈller et al., 1998) and with diets rich in concentrates (Wallace et al., 1981; Dong et al., 1997). Also, in vivo similarn-butyrate proportions were reported for instance in steers either fed a hay±barley-based diet (Eadie et al., 1970) or grass silage together with a concentrate rich in molasses (Moloney et al., 1994). In contrast, lowern -butyrate proportions were reported from hay-based diets in Rusitec (Czerkawski and Breckenridge, 1977; Dong et al., 1997) indicating that elevated contents of highly fermentable carbohydrates frequently favoursn-butyrate production. Compared with the unsupplemented control, with canola oil ciliates and bacteria counts were slightly lower. This can be explained by the inhibitory effect of fatty acids on ciliates and ®bre degrading bacteria (McAllister et al., 1996) and also by the generally lower supply of fermentable matter because a part of the carbohydrates was replaced by lipids. For several reasons ciliate counts are generally lower in Rusitec (Carro et al., 1995; MachmuÈller et al., 1998) than in vivo (Jouany and Ushida, 1999). The protozoal density, however, was repeatedly found to be still high enough to re¯ect dietary effects. Consequently, effects mediated through protozoa might be smaller but nevertheless existing. In order to reduce loss of protozoa by washing out with buffer and together with feed particles, in the present experiment dilution rate was kept rather low (Czerkawski and Breckenridge, 1977).
Lipid supplementation of diets mostly reduces rumen degradation of ®bre (Zinn, 1989; Tesfa, 1992) and, along with that, of organic matter (MachmuÈller et al., 1998) and energy (Jenkins and Fotouhi, 1990). This was not found in the present study at, however, a generally quite low rate of degradation of cell wall carbohydrate fractions, when compared with other experiments, particularly those carried out in vivo. Reasons for this could have been (i) the amylase treatment before NDF and ADF determination which was frequently omitted in other studies (e.g. Carro et al., 1995; Dong et al., 1997); and (ii) the limited access of rumen ¯uid microbes to all sites within the nylon bags as is also the case in situ (Huntington and Givens, 1995). The latter was counterbalanced in the present study as far as possible by the continuous movement of the nylon bags in the system and by the use of a comparably big pore size of 100mm. Compared with control, pH was slightly higher with the canola oil diet, but this was not suf®cient to explain the numerically higher ®bre degradation since pH generally remained within the optimum range (Nagaraja et al., 1997) although still being lower than found by Czerkawski and Breckenridge (1977) with Rusitec.
directly obvious from ®bre disappearance out of the nylon bags. From other investigations controversial results on the effects of canola oil on ruminal ®bre degradation were reported (Tesfa, 1992, 1993; Doreau et al., 1993; Dong et al., 1997). Overall the results indicate a certain and different direct toxic effect of the lipids against the microbes involved in methanogenesis as also found by Dong et al. (1997) using canola oil. Inhibitory effects of unsaturated fatty acids can be expected for methanogens (Nagaraja et al., 1997) and, maybe to a lesser degree, for Gram-positive cellulolytic bacteria (Nagaraja et al., 1997) and ciliates (McAllister et al., 1996) which provide hydrogen as a substrate for the methanogens. This is supported by the slight increase in hydrogen release with the canola oil diet which in turn was far less than the corresponding reduction in hydrogen incorporation in methane (see MachmuÈller et al., 1998).
Less intensively investigated than the use of pure oils is the application of oil provided in a form which partially protects rumen microbes from the fatty acids. This was done in the present study by the use of whole crushed canola seed. This type of processing was repeatedly con®rmed to ful®l this purpose (Murphy et al., 1987; Drochner and Heller, 1996). Nevertheless, in the present study the differences in the effects of canola oil and canola seed were found to be relatively small. At equal pH, concentration of total volatile fatty acids was slightly higher with canola seed but the proportions of the individual main volatile fatty acids remained almost unchanged. The number of ciliates was nearly the same and bacteria count was only slightly higher in the canola seed diet than in the canola oil diet. Furthermore, no major differences in total gas production, methane and hydrogen release and in hydrogen balance occurred. The higher ammonia concentration with whole crushed canola seed most probably resulted from the higher amount of dietary protein degraded and to some extent perhaps also from the higher ruminal degradability of canola protein compared with soybean protein (FAG, 1994) which was supplied at higher proportion in the canola oil diet. The higher protein degradation with canola seed obviously was not compensated by a higher microbial amino acid de novo synthesis. This is also indicated by a higher proportion of short chain iso-fatty acids with canola seed resulting from the fermentation of amino acids (Tamminga and Doreau, 1991) as well as necessary for microbial de novo synthesis of amino acids (Wallace et al., 1997). However, this had no effect on apparent degradation rate of protein and other nutrients except of ether extract which re¯ected the lower accessibility of the oil still incorporated in the seed. Overall, possibly because of its relatively low content of dienoic and polyenoic fatty acids and the limited dietary proportion supplied, the effects of canola oil on rumen fermentation remained small thus preventing a greater differentiation from the effects of canola seed. In a study of Pallister and Smithard (1987) also only modest differences in the effects on rumen fermentation were found when canola oil was supplied as pure oil or as seed.
4.2. Ef®cacy of raw and modi®ed canola lecithins to reduce the oil effects on rumen fermentation
®nding could be that, compared to pure oils, raw lecithins have a higher viscosity and deoiled lecithins furthermore melt at higher temperatures (Pardun, 1988). In the canola lecithin diets, pH of rumen ¯uid was in a similar range as in the canola oil diet whereas total VFA concentration was slightly higher with canola lecithins than with canola oil. The effect was more pronounced with the deoiled (2 mmol lÿ1) and the deoiled/ partially hydrolysed canola lecithin (4 mmol lÿ1) than with the not deoiled lecithins (1 mmol lÿ1). With deoiled lecithins acetate to propionate ratio was also slightly higher indicating that more ®brous constituents may have been fermented (Nagaraja et al., 1997). With increasing dispersibility of the canola lecithins bacteria counts were enhanced, but they were generally higher with canola oil treatment than with most canola lecithins. On the other hand, ciliates numbers were higher in all lecithin diets than with the canola oil diet which may be the reason for the lower bacteria counts found with the lecithins (Jouany and Ushida, 1999). Within the canola lecithin diets, the number of rumen ciliates slightly increased with increasing dispersibility in water but without a further compensatory decrease in bacteria count. The content of residual oil and, inversely related, of phospholipids seems to be less important than dispersibility in this respect as the difference in ciliate count between raw canola lecithin and partially hydrolysed/acetylated canola lecithin illustrates.
One of the major objectives of the present study was to evaluate whether depression in ®bre digestion is alleviated by the use of lecithins, particularly of deoiled or otherwise modi®ed lecithins, instead of pure oils. This was assumed from theoretical considerations based on the assumptions that coating might be one of the factors preventing ®bre digestion (Jenkins, 1993) and that coating is less pronounced with lecithins, particularly when technologically increased in dispersibility. Nevertheless, in the present investigation no difference was obvious in the apparent rate of degradation of ®brous feed constituents between the canola oil and the canola lecithin diets. However, methane depression was less pronounced with the use of canola lecithins instead of canola oil. Furthermore, methane production obviously was more affected by the residual oil content than by the different dispersibility of the lecithins in water as can be seen from the differences in methane release with the deoiled lecithins (CLd, CLd/h) and with partially hydrolysed/
acetylated lecithin (CLh/a). Similar to canola oil also with canola lecithins hydrogen
release behaved largely contrary to methane release. The amount of total fatty acids supplied was similar with canola lecithin and canola oil, but canola lecithins contain slightly more polyenoic fatty acids which affect methanogens (Nagaraja et al., 1997). Therefore, the lower reduction of methane release with lecithin than with canola oil indicates that phospholipids were either not hydrolysed to the same extent or slower as triglycerides (Jenkins et al., 1989) what would also less impair ®bre degrading microbes as discussed in Section 4.1. This is supported by the reduced effects on ciliates and by results from a subsequently carried out in vivo experiment when the use of soy lecithin instead of soy oil increased ®bre digestion (Wettstein et al., 1999). Jenkins et al. (1989) also observed that deoiled soy lecithin did not change ®bre digestion in vitro and Abel-Caines et al. (1998) found a higher rate in NDF degradation with a mixture of soy lecithin and soapstock than with soybean oil.
rumen ammonia concentration. The lower percentage of iso-butyrate and iso-valerate with the lecithin diets is another indicator for a reduced protein degradation since the short chain iso-fatty acids result from the fermentation of amino acids (Tamminga and Doreau, 1991). There might be two major reasons for these favourable effects in protein degradation. Firstly, phospholipids such as lecithins have an amphiphatic behaviour in contrast to triglycerides. This results in a high af®nity to protein (Jenkins et al., 1989). Secondly, phospholipids can promote particle formation of protein (Ono et al., 1996). Lecithin-complexed protein and protein in particulate form both probably are less available to ruminal degradation. Jenkins et al. (1989) showed that there are two fractions within the phospholipids, one being rapidly and one being slowly disappearing within the rumen. The latter ensures that at least part of the lecithins and, consequently, of the associated proteins should reach the small intestine. Similar ®ndings were reported by Jenkins and Fotouhi (1990) with sheep when soy lecithin supplementation of the diets reduced rumen ammonia concentration and increased non-microbial protein ¯ow to the duodenum even at lower protein intake. Modi®cation of canola lecithin had only small further effects on apparent protein degradation.
4.3. Importance of the plant origin of the lecithin for its effects on rumen fermentation
In the present study, deoiled lecithins from canola and soybean origin were investigated (CLdand SLd). Their fatty acid composition differed greatly, particularly with respect to
their content of linoleic acid (Table 1). Linoleic acid is one of the polyunsaturated fatty acids which are known to be more active against ®bre digesting microbes than the monounsaturated fatty acids like oleic acid (Nagaraja et al., 1997). Accordingly canola seed was found to be less adverse to ®bre degradation than sun¯ower seed and linseed (Ossowski, 1999). Ruminal pH and VFA concentration were similar with deoiled canola and soy lecithin, but the acetate to propionate ratio as well as methane release were slightly lower with deoiled soy lecithin than with deoiled canola lecithin, and hydrogen release was correspondingly higher. This indicates that due to the higher content of polyunsaturated fatty in soy lecithin certain rumen microbes, especially methanogens, were more affected. With the deoiled soy lecithin also less ciliates were found than with the deoiled canola lecithin which was expected by the particular susceptibility of the ciliates to linoleic acid (McAllister et al., 1996). On the other hand, bacteria count was compensatorily increased to some extent with the soy lecithin. Soy lecithin decreased crude protein degradation less effectively than canola lecithin but without any difference in the ammonia concentration. Overall the present results suggest that the differences between oil and the respective lecithins could have been more pronounced with products from soybeans instead of canola, particularly when raw lecithins were used.
5. Conclusions
growing ruminants. The use of the canola lecithins reduces the methane-suppressing effects found with the application of the pure canola oil and the canola seed, indicating that, compared to oils, hydrolysis of the phospholipids and associated toxic effects of free fatty acids were lower and possibly ®bre degradation was less adversely affected. These effects were enhanced by some of the technological modi®cations of lecithin. A particularly favourable effect of canola lecithins might be the decrease of the ruminal protein degradation when compared with the pure oil, the seed or when using no additional lipid source at all. This might ®nally increase the supply of metabolisable protein provided that the additional rumen-bypass protein will not be compensated by a lower microbial protein ¯ow. The latter cannot be totally excluded regarding the present results on the microbial counts in rumen ¯uid. In vivo studies have to con®rm these effects before recommendations for the use of lecithins as a feed ingredients in ruminant diets can be developed. Finally, it remains a matter of cost-bene®t calculation whether or not modi®cation of lecithin is carried out before feeding. Particularly deoiling could be a relatively cheap and ef®cient modi®cation.
Acknowledgements
We are grateful to Prof. M. Wanner, Institute of Animal Nutrition and Prof. U. Braun, Department of Internal Veterinary Medicine, both Faculty of Veterinary Medicine of the University of Zurich, for analysing the volatile fatty acids and for placing the ®stulated cow at our disposal, respectively. We also thank B. JoÈrg for technical support, and M. Gebert, H. Gutknecht and A. Rebaud for their assistance in the laboratory.
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