Effect of Arachis Oil on the Conservation of Heavily Wilted Herbage Ensiled in Plastic Containers II.*-Fate of the arachis oil and its effect on the rumen volatile fatty acids of sheep by N. Jackson and B. K. Anderson Department of Agricultural Chemistry, The Queen’s University of Belfast, Belfast, BT9 6BB

(Revised Manuscript received 13 April, 1971)

Arachis oil was added to heavily wilted ryegrass at a mean level of 4.16% of the total ensiled dry matter. Gas-liquid chromatography of the lipid extract was used to investigate possible chemical changes in the ensiled oil. The data obtained suggest that little degradation of the acids occurred. The percentage of linolenic acid increased and this was accompanied by a decrease in the percentage of linoleic acid and oleic acid. The presence of the oil decreased the volatile fatty acid content of the silage. There were no significant differences in the pH or the proportion of volatile fatty acids present in the rumen liquor of sheep fed the oil-treated silage compared with those in the rumen liquor of sheep fed control silage.

Introduction IN silage, especially that made from well fertilised grass, nitrogen is less likely to be a limiting factor than energy when feeding for meat or milk production. Any deficiency in dietary energy is usually made up by the use of a cereal or a high-cereal concentrate.

Arachis oil is a high-energy source and has been widely used in the diets of poultry, especially in compounding high- energy broiler diets. Jackson & Brown1 carried out an experiment to investigate the possible use of arachis oil as an energy supplement for ruminants, the oil being added to the herbage at the time of ensiling. Jackson2 has recently carried out an experiment in which arachis oil was added to herbage at ensiling to provide additional digestible energy. The addition of the arachis oil to the grass resulted in a reduc- tion in the loss of digestible nutrients in the silo. Digesti- bility data, metabolisable energy (ME) determinations with sheep and a voluntary intake study indicate that addition of the oil had a beneficial effect on the voluntary energy intake and utilisation. The present study was undertaken in order to obtain information regarding the fate of the added oil during storage in sealed silos and the effect of the silage on the rumen volatile fatty acids of sheep.

In the ruminant animal, dietary glycerides are hydrolysed to fatty acids and glycerol by the rumen micro-organisms.3 The reducing conditions present in the rumen result in the hydrogenation of the unsaturated fatty acids.*-6 The presence of unsaturated fatty acids in the rumen, and to a lesser extent saturated fatty acids, results in a depression in methane

If arachis oil remains relatively unmodified in the silo then it could lead to a depression of methane energy production similar to that obtained when vegetable oil or fatty acids are fed or infused with dried grass or ha^.^-^

Experimental The second (September) cut of a perennial ryegrass sward

L. perenne) was wilted to approximately 40% dry matter content. Silos 1 and 4 were filled directly while arachis oil was added at a level of 4.26% of the total dry matter in silo 2 and 4-06 % of the total dry matter in silo 3. The herbage was sealed in the silos by enclosing it in polyethylene film. The *Part I: Preceding paper

seams were heat-sealed and the seals then covered with a PVC waterproof adhesive tape. The silos were opened after 4 months.

Lipids were extracted from undried samples of the herbage ensiled and the corresponding silages with chloroform- methanol using the method of Folch et ~ 1 . 1 ~ for animal tissues. Czerkawskill found that this extraction procedure followed by saponification gave a somewhat smaller yield of total fatty acids than direct saponification of the grass, although the composition of the total fatty acids was the same by both methods. The lipids in the extract were saponified prior to esterification using a boron trifluoride-methanol reagent. The resulting fatty acid methyl esters were extracted with hexane and determined by gas-liquid chromatography, using a column packed with 10% PEGA on 100-120 mesh Celite.

Total volatile acids in the silages were determined by Woodman’sI2 method and individual volatile fatty acids were determined by gas-liquid chromatography. The acids in the steam distillate obtained by WoodmanV2 method were neutralised, the solution was concentrated by evaporation, and then acidified. The sample was injected directly onto the column. The columns were packed with 15% Tween 80 (polyoxyethylene sorbitan mono-oleate) coated on 70-80 mesh ‘Embacal’ and used in conjunction with a Pye Series 104 Dual Flame Ionization Chromatograph. Nitrogen was used as the carrier gas and the operating temperature was 105”c. The detector response was quantitated with a standard solution of volatile fatty acids of similar composition to the concentrated steam distillate. Lactic acid was determined by the method of Barker & Summerson13 and water-soluble carbohydrates by the method of Deriaz.l*

Each silage was also fed to three fistulated sheep in order to assess the effect of the added oil on the pH value, total and individual volatile fatty acid (VFA) proportions in the rumen liquor. The fistulated sheep were fed the diets for 10 days in 2 equal feeds, at 9.00 a.m. and 9.00 p.m. The diets were offered at a level just below the maximum intake level to prevent any of the feed being refused.

The rumen contents were sampled through the cannulae every 2 h over the 12 h feeding period on the 10th day of feeding the diet. The samples of rumen liquor were strained through muslin and the p H values of the filtrate were deter- mined immediately. The rumen liquor was treated with 2%

J. Sci. Fd Agric., 1971, Vol. 22, August

Jackson & Anderson: Arachis Oil and Conservation of Heavily Wilted Silage. 11 425

of its volume of saturated mercuric chloride solution as a preservative. The 6 samples of rumen liquor for each sheep were bulked for total and individual VFA determinations. The total VFA content of the rumen liquor was determined using the method described by Annison.15 For the determina- tion of the individual VFA values the rumen liquor was treated with an equal volume of N-HzSO~ saturated with magnesium sulphate. The mixture was allowed to stand for 10 min and the individual VFA values were determined by gas-liquid chromatography by the procedure described above for the silage VFA values.

Results and Discussion The data for the percentage chloroform-methanol extract

are presented in Table I, together with the petroleum ether extract b.p. (4@60°c), dry matter and water-soluble carbo- hydrate percentages. The edible control silage contained 3 % less chloroform-methanol extract than the ensiled grass while ensiling resulted in a 5.5% fall in the chloroform- methanol content of the edible oil-treated material. The chloroform-methanol extract of the grass comprised, on average, 19.6 % unsaponifiable material. Unsaponifiable material comprised, on average, 22.3% and 10.7% of the chloroform-methanol extract of the control and oil-treated silages respectively.

The fatty acid composition of the materials ensiled and of the resultant silages are presented in Table 11. The analysis of the grass for silos 1 and 4 was the mean of two bulked samples. The analyses of the material in silos 2 and 3 were

computed from the grass analysis and the composition of the fatty acids in the arachis oil. The fatty acid composition of the arachis oil was as follows: stearic acid (18 : 0), 1.7%; oleic acid (18 : l), 57.5%; linoleic acid (18 : 2), 17.3%; palmitic acid (16 : 0), 9.8%; and others, 4.1 %.

The composition of the fatty acids in the grass was similar to that found by other workers.le In the grass the predomi- nant fatty acid was linolenic acid (18 : 3) which composed 51 % of the total fatty acids while palmitic acid constituted 20% of the total fatty acids. The percentage of linolenic acid was lower and of palmitic acid higher than that found for perennial ryegrass by Czerkawski'l although in the present work the perennial ryegrass did not come from a pure sward. The mean ratio of linoleic to linolenic acid was 0-225 both for the grass ensiled and the corresponding silage and was rather higher than the values of 0.18 to 0.20 reported by Czerkawski.11 Shorlandl7 reported that lipids in the leaves of ryegrass comprised 3.4 % free acids, 25.3 % triglycerides and 71.3 % galactose-glyceride esters. He found the predominant acid in each group to be linolenic acid (18 : 3) and this comprised 88 % of the galactose-lipid fraction. The presence of a higher proportion of linolenic acid in grass is of particular interest since Czerkawski et al.7 have shown this acid to be more effective than oleic or linoleic acid in depres- sing methane production in the rumen. The fatty acid composition of the ensiled material was considerably modi- fied by the addition of the arachis oil and oleic acid became the predominant acid. The data do not indicate any hydro- genation of linolenic acid in the silages. The figures suggest

TABLE I Ether extract, chloroform-ethanol extract, water-soluble carbohydrate and

dry matter contents of ensiled material and edible silage dry matter ~

Control silages Oil-treated silages

Silo 1 Silo 4 Silo 2 Silo 3

Grass Silage Grass Silage Grass Silage Grass Silage +oil +oil

Ether extract, % 1.77 2.69 2.18 3.05 7.02 7.21 5.97 6.59 Chloroform-methanol extract, % 4.19 4.29 4.19 3.85 8.43 8.24 8.40 7.67 Water-soluble carbohydrate,

as glucose, % 17.20 4.08 15.68 3.26 17-22 5.02 17.99 3.66 Dry matter, % 38.78 35.86 40.14 37.94 41.65 39.43 43.48 41.60

TABLE I1 Fatty acid composition of the material ensiled and the silage, % of dry matter

Control silages Oil-treated silages

Silo 1 Silo 4 Silo 2 Silo 3

Grass Silage Grass Silage Grass Silage Grass Silage +oil +oil

20 : 4 Arachidonic 20 : 0 Arachidic 18 : 3 Linolenic 18 : 2 Linoleic 18 : 1 Oleic 18 : 0 Stearic 16 : 1 Palmitoleic 16 : 0 Palmitic 16 : 0 Isopalmitic Others Total fatty acids* S.E. of total fatty acid determination*

Ll . 1.20 0.27 0.10 0.04 0.06 0.47 0.10 0.10 2.35

+0.35

0.03 0.06 1.34 0.35 0.19 0.08 0.09 0.74 0.06 0.14 3.08

rt0.64

tr. 1.20 0.27 0.10 0.04 0.06 0.47 0.10 0.10 2.35

1 0 . 3 5

1.41 0-27 0.09 0.03 0.06 0.49 0.04 0.08 2.47

rt0.38

- 0.01 i.io 0.97 2.53 0.11 0.06 0.83 0.10 0.25 5.96

1 0 . 1 1

- tr.

1.47 0.76 1.79 0.11 0.07 0.85 0.08 0.14 5.27

1 0 . 2 8

1.21 0.98 2.43 0.11 0.05 0.89 0.09 0.29 6.05

5 0 . 3 5

0.01 0.04 1.33 0.78 2.05 0.12 0.09 0.87 0.08 0.16 5-53

rt0.35

* Based on analysis of three sub-samples of bulked material J. ISci. Fd Agric., 1971, Vol. 22, August

426 Jackson & Anderson: Arachis Oil and Conservation of Heavily Wilted Silage. I1

that the percentage of linolenic acid increased as a result of the ensilage process and this appeared to be accompanied by a decrease in the percentage of linoleic and oleic acid in the oil-treated silages. The grass was stored in a deep freeze (-lSoc) and the lipids were extracted without drying; some of the observed changes in fatty acid composition could possibly have occurred during the cold storage period and not in the silo. Thus the fatty acid composition of the silages shows no evidence of major hydrogenation occurring under the anaerobic ensilage conditions nor does it indicate any breakdown of the long-chain acids to shorter-chain acids.

The results for the total volatile fatty acids and percentages of the individual acids present in the silages are presented in Table 111.

The pH value, total VFA and the molar proportions of the individual acids in the rumen liquor of fistulated sheep fed on the two diets are presented in Table IV.

TABLE I11

Total volatile fatty acids in the edible silage dry matter and the percentages of individual volatile acids in the

volatile acids fraction

Control Oil-treated

Silo 1 Silo 4 Silo 2 Silo 3

Total VFA, % in silage

Acetic acid, % in

Propanoic acid, % in

Butanoic acid, % in

Others (as butanoic

dry matter 2.68 1’95 1.72 1.59

VFA fraction 28.9 53.9 91.5 65.8

VFA fraction 2.2 2,5 1.6 1.5

VFA fraction 67.0 41.9 6.3 28.1

acid), % in VFA fraction 1.9 1-7 0.6 4.6

TABLE IV pH, total and the molar proportions of individual volatile fatty acids

in the rumen liquor of sheep fed the silage diets

Control silage*

PH 6.87 Total VFA, mequiv./100 ml 8.73 Acetic acid, % 71.9 Propionic acid, % 18.1 Isobutyric acid, % 0.9 Butyric acid, % 6.3 Isovaleric acid, % 1.7 Valeric acid, % 1.1

Arachis S.E. of oil-treated treatment

silage* mean

6.67 h0.05 9.09 A0.12

71.6 k0 .94 18.4 1 1 . 2 7 0.9 h0 .12 6.1 k0 .26 2.0 &0.18 1.0 1t0.31

* Three fistulated sheep fed each silage

The pH values and contents of lactic acid and volatile acids in the silages (Table 111) suggest that although the presence of the added lipid may have modified the fermentation process in the silage, as evidenced by the decreased production of total volatile fatty acids and the increased proportion of acetic acid, there was no major breakdown in the rumen of the long- chain acids in the added oil, to short-chain volatile fatty acids. The present study did not indicate what proportion, if any, of the added triglyceride was hydrolysed to glycerol and free fatty acids. If this had been appreciable then increased volatile fatty acid formation from free glycerol and reduced pH would have been expected. The data for soluble carbo- hydrates2 in the silage (Table I) do not suggest that fermenta- tion of these was inhibited by the presence of the oil during the ensilage process.

There were no significant differences in the pH value of the rumen liquor of sheep fed the oil-treated silage diets compared with control silage. Total VFA concentration was not significantly affected nor was the major proportion of any of the individual VFA values. There was no evidence of the effect found by Garton et aL3 that the presence of glycerides in the rumen contents led to an enhanced formation of steam- volatile fatty acids, owing to the fermentation of free glycerol. x

The results suggest that when arachis oil is added to grass and the material is ensiled with an efficient exclusion of oxygen, there is no significant alteration in the chemical nature of the added oil during the ensilage period. This would indicate that arachis oil, and possibly other vegetable oils, may be added to grass at the time of ensiling as an addi- tional source of digestible energy.

Acknowledgment The authors thank Mr. C . Black for technical assistance.

References 1 . 2. 3.

4.

5.

6.

7.

8.

9.

10.

11. 12. 13.

14. 15. 16.

17. 18.

Jackson, N., & Brown, W. O., J. Br. Grassld Sac., 1967,22,214 Jackson, N., J. Sci. FdAgric., 1971,22,419 Garton, G. A., Lough, A. K., & Vioque, E., J . gen. Microbiol., 1961.25.215 Shodand, F. B., Weenink, R. O., &Johns, A. T., Nature, Lond., 1955, 175, 1129 Reiser, R., & Reddy, H. G. R., J. Am. Oil Chem. Soc., 1956, 1?. 1 5 5 --, - -_ Shorland, F. B., Weenink, R. O., Johns, A. T., & McDonald, 1. R. C.. Biochem. J.. 1957.67. 328 Czerkawski, J. W., Blaxter, K. L., & Wainman, F. W., Bv. J. Nutv., 1966,20, 349 Czerkawski, J. W., Blaxter, K. L., & Wainman, F. W., Br. J. Nutr., 1966, 20, 485 Clapperton, J. L., & Czerkawski, J. W., Br. J. Nutr., 1969, 23, 813 Folch, J., Lees, M., & Sloane-Stanley, G. H., J. bid. Chem., 1957, 226,497 Czerkawski, J. W., Bv. J . Nutr., 1967, 21, 599 Woodman, H. E., J. agric. Sci., Camb., 1925, 15, 343 Barker, S. B., & Summerson, W. H., J. biol. Chem., 1941, 138, 535 Deriaz, R. E., J. Sci. Fd Agric., 1961, 12, 152 Annison, E. F., Biochem. J., 1954, 57, 400 Hilditch, T. P., & Williams, P. N., ‘The chemical constitution of natural fats’, (4th edn) 1964 (London: Chapman & Hall) Shorland, F. B., J. Sci. Fd Agric., 1961, 12, 39 Johns, A. T., N.Z. JISci. Technol., 1953, 35A, 262

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