The effects of level of fish oil inclusion in the diet
on rumen digestion and fermentation parameters
in cattle offered grass silage based diets
T.W.J. Keady
a,*, C.S. Mayne
a,baAgricultural Research Institute of Northern Ireland, Hillsborough, Co. Down BT26 6DR, UK bDepartment of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, UK
Received 7 December 1998; received in revised form 26 April 1999; accepted 19 May 1999
Abstract
A partially balanced changeover design experiment was undertaken to evaluate the effects of level of fish oil inclusion in the diet on rumen fermentation parameters and digestion with 10 beef cattle offered grass silage ad libitum as the basal forage supplemented with 5 kg concentrates headÿ1dayÿ1. Concentrates were prepared to provide either 0 (T0), 150 (T150), 300 (T300) or
450 g (T450) fish oil (Fish Industries, Killybegs, Co. Donegal, Ireland) or 300 g (T300B) fish oil premix (J. Bibby Agriculture) per head per day. The concentrates were formulated to have similar concentrations of crude protein, effective rumen degradable protein, digestible undegradable protein and starch. The dry matter (DM), pH and ammonia nitrogen (N) concentrations of the silage were 194 g kgÿ1, 3.98 and 94 g kgÿ1 N, respectively. Level or source of fish oil did not alter
(P> 0.05) the disappearance of DM, neutral detergent fibre or acid detergent fibre after 12 or 24 h rumen incubation intervals. Increasing the level of fish oil increased rumen ammonia concentration (P< 0.001) but did not alter (P> 0.05) rumen pH or the molar concentrations of the volatile fatty acids. The fish oil premix decreased rumen ammonia concentration (P< 0.001), the molar concentrations of acetate (P < 0.05), the acetate : propionate (P< 0.05), acetate + butyrate/ propionate (P< 0.01) and non-glucogenic (P< 0.05) ratios and increased the molar concentration of propionate (P< 0.01). It is concluded that changes in rumen fermentation parameters do not account for the depressions in milk butterfat content with fish oil inclusion observed in a concurrent production study in which lactating dairy cows were offered similar diets to those used in the present study. Furthermore, the changes in rumen fermentation parameters with inclusion of a fish oil premix are probably associated with the carrier or the source of fish oil used in that product. #1999 Elsevier Science B.V. All rights reserved.
Keywords: Cattle; Fish oil; Rumen; Fermentation; Digestibility
*Corresponding author. Tel.: +44-1846-682484; fax: +44-1846-689594
E-mail address: [email protected] (T.W.J. Keady)
1. Introduction
Given the current constraints on milk fat production within the European Union (EC), there is considerable interest in developing strategies to reduce milk butterfat content whilst maintaining milk output. It is generally accepted that inclusion of fish oil in the diet depresses butterfat content of milk when animals are offered hay (Beitz and Davis, 1964; Nicholson and Sutton, 1971; Brumby et al., 1972) or maize silage (Chilliard and Doreau, 1997) as the basal forage. More recently, Keady et al. (1999a) concluded that increasing the level of fish oil in the diet decreased milk fat content of dairy cows offered grass silage as the basal diet, regardless of the level of concentrate supplementation. Possible reasons for the decreased milk fat content with fish oil supplementation include inhibition of de-novo fatty acid synthesis and mammary gland uptake of plasma fatty acids (Brumby et al., 1972), inhibition of lipoprotein lipase activity (Storry et al., 1969) or mammary acetyl-CoA carboxylase (Moore and Steele, 1968), or as a result oftransfatty acid production. It is also possible that fish oil inclusion in the diet may alter the rumen environment, consequently decreasing fibre digestion and the ratio of lipogenic : gluco-genic fatty acids. Several previous studies have shown that fish oil supplementation of cows offered hay (Nicholson and Sutton, 1971; Brumby et al., 1972; Storry et al., 1974) or maize silage (Chilliard and Doreau, 1997) as the basal forage, resulted in a reduction in the ratio of lipogenic to glucogenic fatty acids in the rumen. However, in these studies the fish oil was offered in one feed and samples of rumen fluid were collected on a few occasions, within a maximum period of 10 h post feeding.
Given the paucity of experimental data on the effects of fish oil supplementation on rumen digestion and fermentation patterns of cattle offered grass silage based diets the present study was initiated to determine if the effects of fish oil inclusion on milk fat concentration in the concurrent study (Keady et al., 1999a) were mediated through changes in rumen fermentation parameters. The effects of fish oil inclusion on the disappearance of dry matter (DM), neutral detergent fibre (NDF) and acid detergent fibre (ADF) in the rumen were also examined.
2. Material and methods
2.1. Silage
Grass silage was produced from herbage harvested from the primary growth of predominantly perennial ryegrass swards which had received 7.6 m3cattle slurry and 127, 17.5 and 35 kg haÿ1of nitrogen (N), P
2O5and K2O, respectively. It was mown between 4 and 7 June using a mower fitted with a V-spoke grass conditioner (Taarup, Model 307) and harvested after a wilting period of 24 h using a precision chop forage harvester (Reco-Mengele, Model SH40N). At ensiling the herbage was treated with an inoculant (Ecosyl, Zeneca Bio Products) which was applied through a pump applicator and discharged into the auger chamber of the harvester at the rate of 2.98 l tÿ1
herbage. During filling, the silo was consolidated between loads by rolling with an industrial loader and for a further 60 min after filling was completed. Following consolidation, two
polythene sheets were used to seal the silo. The entire surface was then weighed down with a layer of tyres.
During the feeding period, silage was removed from the silos with a shear grab in blocks containing approximately 0.5 m3.
2.2. Concentrate
Five concentrates were formulated containing different levels and sources of fish oil. Initially three concentrates were prepared containing no fish oil (Control) and fish oil (FO) (Fish Industries, Killybegs, Co. Donegal, Ireland) at 90 kg tÿ1
or a commercial fish oil premix (FOP) (J. Bibby Agriculture) at 120 kg tÿ1
. Two further concentrates were prepared by mixing the control and FO concentrates in different proportions. The concentrates were formulated to supply either 0 (T0), 150 (T150), 300 (T300) or 450 g (T450) fish oil animalÿ1
dayÿ1
or 300 g (T300B) animalÿ1 dayÿ1
of the alternative commercial fish oil premix, when offered at 5 kg headÿ1
dayÿ1
. The concentrates were formulated to have similar concentrations of crude protein (CP), effective rumen degradable protein (ERDP) and digestible undegradable protein (DUP) using AFRC (1993) published values for individual feed ingredients and starch. Concentrate formulations are presented in Table 1. Cereals and pelleted ingredients were ground through a 3 mm screen before mixing and pelleting through a 12 mm die.
2.3. Animals and management
The diets were offered to 10 mature steers fitted with rumen fistulae in a partially balanced, changeover design experiment consisting of three 4-week periods giving a total of six animals per treatment. During changeover, the concentrate treatments were
Table 1
Ingredient composition of concentrates (g kgÿ1fresh weight)
Concentrate
Molassed sugar beet pulp 100 95 100
gradually introduced during a 3-day period. Silage was offered once daily at 09:00 in sufficient quantities to allow a refusal of 50 to 100 g kgÿ1
intake. The concentrates were offered in two equal feeds at 05:00 and 16:00 h. The animals were housed in individual tie-up stalls. Individual intakes of silage and concentrates were recorded daily for the duration of the study, with the last week of each period being the main recording interval.
2.4. Measurements
Samples of offered and refused silage were retained daily for oven DM (858C) analysis. The dried samples of offered silage were bulked weekly and analysed for ADF, NDF, ash and acid detergent insoluble nitrogen (ADIN) concentrations. Fresh samples of offered silage were obtained twice weekly for the determination of pH, buffering capacity and concentrations of toluene DM, CP, ammonia N, ethanol, propanol, acetate, propionate, valerate, butyrate and lactate.
A sample of each concentrate type was retained weekly for the determination of oven DM (1008C), CP, ash, ether extract (EE), NDF, water soluble carbohydrates (WSC), gross energy, starch, NDF cellulose digestibility and ADIN.
2.5. Rumen digestion study
The effects of fish oil supplementation on rumen digestion of hay were determined by the polyester bag method as described by Mehrez and érskov (1977). Eight polyester bags (pore size = 1600mm2) containing the equivalent of 5 g DM of fresh chopped hay
(ADF and NDF concentrations of 420 and 722 g kgÿ1DM, respectively) were suspended
in the rumen of the steers fitted with rumen fistulae on day 26 of each sampling period. Four polyester bags were removed after the 12 and 24 h incubation periods, respectively.
2.6. Rumen fermentation study
On day 28 of each period samples of rumen liquor were collected via the fistulae at 05:00, 06:00, 07:00, 09:00, 10:00, 13:00, 16:00, 17:00, 19:00, 22:00 and 05:00 h representing 0, 1, 2, 4, 5, 8, 11, 12, 14, 17 and 24 h after the morning feed, respectively. The samples of rumen liquor were obtained as described by Keady and Steen (1996) and were analysed for pH, ammonia N, acetate, propionate, butyrate and valerate as described by Keady et al. (1994). Other methods of chemical analysis of the silages and concentrates were as described by Keady et al. (1998).
2.7. Statistical analysis
Data on food intake, rumen digestion and fermentation parameters were analysed as partially balanced changeover design experiment using the residual maximum likelihood (REML) directive in the GENSTAT 5 statistical software package. For food intake and rumen digestion data, the effect of oil was investigated using the REML, while for the rumen fermentation data, REML was used to investigate the effects of oil, time and the
interaction between oil and time. Linear and quadratic contrasts were calculated for the four levels of fish oil. Differences between treatments were tested using the studentt-test.
3. Results
3.1. Chemical composition of the silages and concentrates
The chemical composition of the silage as fed is presented in Table 2. The silage was well preserved as measured by its pH and low concentrations of ammonia N and butyrate. The chemical composition of the concentrates at feeding is presented in Table 3. The concentrates had similar concentrations of DM, CP and starch. Increasing the level of fish oil in the concentrate decreased the concentrations of WSC and ash, and increased the concentrations of ADIN, EE and gross energy. Relative to T300, T300B had lower concentrations of WSC and gross energy and higher concentrations of ADIN and ash. The fatty acid profiles of the fish oil, fish oil premix and the concentrates at feeding are presented in Table 4. Increasing the level of fish oil inclusion in the concentrates increased the concentrations of C12:0, C14:0, C18:0, C20:0, C20:4w3, C20:4w6, C22.0, C22:1, C20:5w3, C24:0, C24:1w9and C22:6w3and decreased the concentrations of C18:2, and C18:3in total fat. Relative to T300, T300B increased the concentrations of C18:2and C20:5w3and decreased the concentrations of C14:0, C20:0and C22:6w3.
3.2. Food intake
The effects of fish oil treatment on silage and total DM intake are presented in Table 5. Increasing the level of fish oil supplementation tended (P> 0.05) to decrease silage and total DM intakes. There were no significant (P> 0.05) linear or quadratic contrasts across
Table 2
Chemical composition of the silage at feeding
Dry matter (alcohol corrected toluene) (g kgÿ1) 194
pH 3.98
Buffering capacity (mEquiv kgÿ1DM) 1045
Composition of DM (g kgÿ1)
Crude protein 135
Ammonia nitrogen (g kgÿ1total N) 94
Ethanol 9.2
Propanol 1.2
Acetate 25
Propionate 0.2
Butyrate 0.4
Valerate 0.5
Lactate 75
ADF 403
NDF 664
ADIN 4.4
Table 3
Chemical composition of the concentrates at feeding
Concentrate
T0 T150 T300 T450 T300B
Chemical composition
Dry matter (g kgÿ1) 861 864 867 870 862
Composition of dry matter (g kgÿ1 )
Crude protein 219 217 215 213 218
Water soluble carbohydrate 101 98.7 96.3 94 90
Neutral detergent fibre 308 307 305 304 332
Starch 307 301 296 290 303
NDF cellulose digestibility 824 811 797 784 777
Acid detergent insoluble nitrogen 2.94 3.13 3.32 3.52 4.54
Ash 84 83.3 82.6 82 86
Ether extract 28 54 79 105 81
Gross energy (MJ kgÿ1DM) 17.87 18.33 18.80 19.26 18.52
Table 4
Fatty acid profile of the fish oils and of the concentrates at feeding
Fatty acid
the four levels of fish oil. Source of fish oil did not alter (P> 0.05) either silage or total DM intakes.
3.3. Rumen fermentation parameters
The effects of fish oil treatment on rumen fermentation parameters are presented in Table 5. Relative to T0 and T300, T450 increased (P< 0.05) the concentrations of rumen ammonia. T450 decreased (P< 0.05) the non-glucogenic ratio relative to T300. Other than for the linear effect of ammonia (P< 0.05) there were no significant (P> 0.05) linear or quadratic contrasts across the four levels of fish oil. Level of fish oil did not significantly (P> 0.05) alter rumen pH, the concentrations of ammonia, total volatile fatty acids (VFA), ethanol or propanol, or the molar concentrations of acetate, butyrate, propionate or valerate. Relative to T300, T300B increased the concentrations of ammonia (P< 0.001) and the molar proportion of propionate (P< 0.01) and decreased the molar proportions of acetate (P< 0.01) and the acetate + butyrate/propionate (P< 0.01) acetate/ propionate (P< 0.01) and the non-glucogenic ratios (P< 0.01).
All the rumen fermentation variables determined in the present study were significantly (P< 0.05 or greater) altered by time of sampling.
There was a significant fish oil treatment by sampling time interaction (P< 0.01) for the concentration of rumen ammonia (Fig. 1). Treatment T300B significantly increased the concentrations of ammonia at 1 and 2 h relative to the other treatments and at 4 h
Table 5
Effect of fish oil treatment on food intake and rumen fermentation parameters
Treatment s.e.m. Significance
T0 T150 T300 T450 T300B Treatmenta Time Treatment
time
Food intake (kg DM dayÿ1 )
Silage 7.50 6.80 7.21 6.76 6.95 0.251 NS
Total 12.14 11.47 11.88 11.45 11.59 0.249 NS
Rumen fermentation
pH 6.17 6.11 6.14 6.14 6.21 0.035 NS *** NS
Ammonia (m mol lÿ1) 6.82a 7.72ab 7.22a 8.46bc 8.97c 0.364 *** *** **
Ethanol (m mol lÿ1) 0.51 0.61 0.67 0.69 0.82 0.152 NS *** NS
Propanol (m mol lÿ1) 0.00 0.06 0.00 0.04 0.00 0.021 NS *** NS
Total VFA (m mol lÿ1) 114 110 109 110 105 3.4 NS *** NS
Molar concentrations of VFAs (mmol/mol total VFAs)
Acetate (Ac) 667b 662ab 672b 652ab 641a 7.8 * *** NS
Propionate (Pr) 184a 185a 182a 195ab 203b 5.8 ** *** NS
Butyrate (But) 124 127 123 126 126 4.7 NS *** NS
Valerate (Va) 24 26 24 27 30 3.2 NS * NS
Ac/Pr 3.76b 3.75b 3.86b 3.46ab 3.24a 0.169 * *** NS
Ac + But/Pr 4.45b 4.45b 4.55b 4.12ab 3.87a 0.183 ** *** NS
Non-glucogenic ratio 4.64bc 4.62bc 4.72c 4.25ab 4.07a 0.159 *** *** NS
aOther than for the linear effect of ammonia (P< 0.05), there were no significant (P> 0.05) linear or quadratic
relative to the control. At 8 h T150 increased ammonia concentrations relative to T300. Relative to the control, rumen ammonia concentration was increased for T300B at 12 h and for T450 at 12 and 14 h after the morning concentrate feed. Treatment had no effect (P> 0.05) on rumen ammonia concentrations at 0, 5, 11, 17 or 24 h post feeding. There were no treatment by time interactions (P> 0.05) for rumen pH, the molar concentrations of acetate, propionate, butyrate, the ratios of acetate : propionate, acetate + butyrate/ propionate and non-glucogenic ratios or the concentrations of ethanol or propanol.
The effects of fish oil treatment on the disappearance of DM, ADF and NDF of hay incubated in the rumen for 12 and 24 h are presented in Table 6. Fish oil treatment did not alter (P> 0.05) the disappearance of DM, ADF or NDF following 12 and 24 h incubation periods, respectively. There were no significant (P> 0.05) linear or quadratic contrasts across the four levels of fish oil.
Fig. 1. The effects of fish oil treatment on rumen ammonia concentration (Control ±&± 150 g fish oil/cow/day ±^± 300 g fish oil/cow/day ±~± 450 g fish oil/cow/day ±$± 300 g fish oil/cow/day from premix ±*±).
Table 6
The effects of fish oil treatment on the proportion of hay disappearance in the rumena
Incubation period (h)
Treatment s.e.m. Significance
T0 T150 T300 T450 T300B
Dry matter 12 0.189 0.180 0.180 0.186 0.192 0.0102 NS
24 0.380 0.363 0.366 0.385 0.375 0.0185 NS
Acid detergent fibre 12 0.067 0.073 0.071 0.082 0.098 0.0183 NS
24 0.259 0.258 0.255 0.278 0.274 0.0241 NS
Neutral detergent fibre 12 0.048 0.041 0.030 0.056 0.053 0.0112 NS
24 0.240 0.227 0.222 0.257 0.247 0.0229 NS
aThere were no significant (P> 0.05) linear or quadratic contrasts across the four levels of fish oil.
4. Discussion
In a concurrent study (Keady et al., 1999a) inclusion of 450 g fish oil cowÿ1 dayÿ1
in the diet of lactating dairy cattle altered milk composition by decreasing the concentrations of fat and protein by 15 and 3.86 g kgÿ1
, respectively. The present study was undertaken to elucidate if the mode of action of fish oil was occurring in the rumen, due to changes either in fermentation parameters or digestibility. In the present study concentrates accounted for approximately 0.60 of the total diet which is similar to the mean of the two concentrate treatments offered in the concurrent study (Keady et al., 1999a) in which there were no fish oil by concentrate feed level interactions for milk composition. As in the concurrent study the concentrates were formulated to contain similar concentrations of CP, starch, ERDP, DUP, WSC and sugars. The ERDP and DUP concentrations were based on AFRC (1993) published values.
4.1. Effects of fish oil on silage intake
Although fish oil treatment did not significantly decrease silage intake, fish oil inclusion resulted in similar proportional decreases in intake as occurred at the higher level of concentrate supplementation in the concurrent study (Keady et al., 1999a). Also Wonsil et al. (1994), Chilliard and Doreau (1997) and Cant et al. (1997) reported reductions in food intake due to fish oil inclusion in the diet. Furthermore, the absence of an effect of fish oil type on silage DM intake in the present study is similar to the results obtained by Keady et al. (1999a). The decrease in silage intake recorded in the present study with fish oil supplementation may be due to a metabolic control mechanism related to the effect of some fatty acids of fish oil on biohydrogenation in the rumen as suggested by Doreau and Chilliard (1997) rather than to a negative effect on rumen function, as fish oil inclusion did not affect rumen fermentation patterns or digestion of fibre fractions, which have been identified as major factors affecting silage intake (Steen et al., 1998).
4.2. Effects of fish oil on rumen digestion
levels and type of fish oil supplementation had little effect on bacteria or protozoa growth. Dong et al. (1994) observed no effect of including cod liver oil at levels up to 0.10 of total DM on total or cellulolytic bacterial numbers in an artificial fermenter containing forage or grain based diets.
4.3. Effects of fish oil on rumen fermentation
Fish oil inclusion had no effect on the composition of fatty acid in the rumen liquor in accord with the results of Beitz and Davis (1964); Doreau (1992) and Wonsil et al. (1994). However Nicholson and Sutton (1971), Brumby et al. (1972), Sutton et al. (1975) and Doreau and Chilliard (1997) concluded that inclusion of fish oil in the diet decreased the molar concentration of acetate and increased the molar concentration of propionate. The absence of any effect of fish oil on rumen volatile fatty acid concentrations may be associated with the sampling procedure employed in the present study, i.e., 11 samples being taken during the 24 h sampling period. In previous studies, (Beitz and Davis, 1964; Brumby et al., 1972; Nicholson and Sutton, 1971; Storry et al., 1974; Sutton et al., 1975; Doreau, 1992; Wonsil et al., 1994; Doreau and Chilliard, 1997) normally only 1 to 3 samples of rumen liquor were obtained within the first 10 h after feeding. In the present study increasing the level of fish oil tended to increase propionate concentrations at 1, 5 and 14 h after feeding but had no effect on propionate concentrations at the other sampling times. Secondly in the present study the fish oil was offered in two equal feeds per day, reducing total oil intake at any one period and consequently reducing potential changes in rumen fermentation pattern. For example, Doreau and Chilliard (1997) offered fish oil in one feed daily and concluded that the inclusion of 200 g fish oil had no effect on rumen fermentation patterns whereas inclusion of 400 g fish oil in one feed reduced the molar proportions of acetate and increased the proportions of propionate. In the present study the maximum level of fish oil offered at any one time was 225 g. Finally grass silage formed the basal forage in the present study whereas in previous studies the basal diet consisted of either hay (Beitz and Davis, 1964; Brumby et al., 1972; Nicholson and Sutton, 1971; Storry et al., 1974; Sutton et al., 1975) or maize silage (Doreau, 1992; Wonsil et al., 1994; Doreau and Chilliard, 1997). Grass silage has a major impact on rumen fermentation parameters. Keady et al. (1999b) offered three grass silages supplemented with concentrates varying in starch content from 17 to 304 g kgÿ1
DM to dairy cows and concluded that relative to concentrate type, silage type had a greater impact on rumen fermentation patterns. Furthermore the rumen fermentation pattern measured at a particular time depends not only on silage composition but also on rate of eating (Offer and Percival, 1998). Although feeding behaviour was not measured in the present study, in the concurrent study Keady et al. (1999a) concluded that as the level of fish oil increased silage intake decreased from 6 to 24 h after the a.m. feed.
It is interesting to note that in the present study the fish oil premix significantly decreased the molar proportions of acetate and increased the proportions of propionate relative to the other fish oil source. These changes in rumen VFA concentrations are possibly associated with the carrier used in this product, namely palm kernel, and/or its fatty acid profile. The fish oil premix had a greater proportion of the longer chain fatty acids (i.e. C20:0and greater) which may have resulted in a modification of the ruminal
microbial ecosystem. Also the fish oil premix resulted in higher intakes of unsaturated oil which are prone to cause changes in the rumen environment.
It is concluded that increasing the level of fish oil up to 450 g per day did not alter rate of rumen digestion or fermentation parameters. However, the fish oil premix decreased the ratio of lipogenic to glucogenic precursors. Consequently the depression in milk butterfat content obtained in the concurrent study cannot be attributed to changes in rumen fermentation parameters (Keady et al., 1999a). The depression in milk fat recorded in the concurrent study (Keady et al., 1999a) was probably due firstly to the inhibition of
de-novofatty acid synthesis and mammary uptake of plasma fatty acids which may have occurred given the presence of C20to C22polyunsaturated acids in fish oil and, secondly, to the production oftransfatty acids, particularly transC18:1, which has been shown to depressde-novofatty acid synthesis.
Acknowledgements
The authors wish to thank Mr W. Clews and the staff of the dairy unit and Mr M. Porter and the laboratory staff for their assistance.
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