P R O T E I N P R O T E C T E D F A T F O R R U M I N A N T S II. S E R U M L I P I D S AND L I P O P R O T E I N S 1 ,2,3 F. D. Dryden, J. A. Marchello, L. L. Cuitun and W. H.

Hale
University o f Arizona, Tucson 85721

compared to the other two treatments. The relative percentage of total unsaturated fatty Qualitative and quantitative serum lipid and acids in the serum between the three treatments lipoprotein data were obtained from 11 steers was not significantly different ( P > .05) at fed an 80% concentrate fattening ration any of the four sampling periods. according to the following treatments: (1) Serum low density lipoproteins (LDL) control ration containing 6% formaldehyde concentration (330 mg/100 mg serum) for the treated casein (three steers), (2) basal ration protected safflower oil diet at 168 days of the supplemented with 6% formaldehyde treated trial was approximately fourfold higher (P < casein plus 6% safflower oil (three steers), and .05) than that of the control or safflower oil (3) basal ration supplemented with 12% of a supplemented animals. None of the three formaldehyde treated spray dried casein:saf- treatments had significantly different (P > .05) flower oil homogenate (five steers). levels of high density lipoprotein (LDL) on the The serum lipid level for the steers final sampling period. Generally, oleic and consuming the protected safflower oil diet from linoleic acid were the only major fatty acids of 112 to 168 days of the experiment was the four lipid classes of the serum LDL and significantly greater ( P < .05) than that of HDL lipid that were influenced (P < .05) by either the control steers or those supplemented dietary treatments. with unprotected safflower oil which did not differ ( P > .05). On the final period, steers Introduction consuming the protected safflower oil product Several recent reports in the literature had an average serum lipid level of approximately 930 mg/100 ml serum which was 2.1 discuss the use of a technique developed by times greater than the control animals and 1.7 Scott et al. (1972) and Cook et al. (1970) times greater than the animals supplemented which causes an increase in polyunsaturation of with unprotected safflower oil. There was an milk and body fats of the ruminant by feeding inverse relationship between serum oleic and polyunsaturated oils emulsified with a protein linoleic acid, the former decreased while the which was subsequently treated with formaldelatter increased (P < .05), for the steers hyde (Plowman et al., 1972; Cook et al., 1,972; consuming the protected safflower oil diet as Scott et al., 1972). Faichney et al. (1972) and Cuitun et al. (1975) have evaluated tile performance of steers fed this type of an Arizona Agricultural Experiment Station Technical emulsified product; however, the effect of the enhanced lipid intake caused by feeding this Paper 2243 2This study was supported in part by a grant from type of product on serum lipids and specific Fats and Protein Research Foundation, Inc., Des lipoprotein concentrations has not been deterPlaines, Illinois 3The spray dried casein:safflower oil homogenate mined. Palmquist and Mattus (1973)have was prepared by SmithKline Corp., Philadelphia, reported elevated (P < .05) levels of serum LDL Pennsylvania through the courtesy of Dr. John E. Trei. (density 1.034 to 1.048) in dairy cows fed full 697
JOURNAL OF ANIMAL SCIENCE, vol. 40, no. 4, 1975

Summary

698

DRYDEN ET AL. successive centrifugations, the major lipoprotein classes. Chylomicrons and very low density lipoproteins were recovered at the assumed background serum electrolyte density of 1.007. These data were not included in the results because of the very low concentration of this fraction which did not vary between treatments throughout the experiment. After density adjustment to 1.063/ml with NaBr and centrifugation, the low density lipoproteins (LDL) (density 1.007 to 1.063) were obtained. Further centrifugation at a NaBr adjusted density of 1.21 g/ml yielded the high density lipoproteins (HDL) (density 1.063 to 1.21). These isolation procedures and concentration determinations have been discussed in detail in a previous publication (Dryden et al., 1971). The lipid portion of the whole serum and lipoprotein fractions were extracted with chloroform-methanol (2:1, V/V)and stored in chloroform at -18C until 35 mg aliquots were utilized for thin-layer separation into phospholipids, free fatty acids, glycerides and cholesterol esters. The thin layer plates (20 X 20 cm, .25 mm silica gel F-2544) were developed with a solvent of petroleum ether (30 to 70 C)-diethyl ether-acetic acid (80:10:1, V/N/V). Identification of specific fractions was accomplished by lightly spraying the plates with a .05% (W/N) solution of Rhodamine 6G in ethyl alcohol and viewing under ultraviolet light. A further discussion of these techniques in addition to the methods of esterification and gas-liquid chromatography of the fatty acids has been presented by Marchello et al. (1971). The nested least-squares procedures of Harvey (1960) were utilized in statistical treatment of the data. The fatty acid analysis and lipoprotein concentration comparisons were made on a within fraction basis with the main effects nested therein. Duncan's New Multiple Range Test according to Li (1964) was applied to isolate significant (P < .05) effects. Results and Discussion The serum lipid levels by 28-day intervals for each dietary group are listed in figure 1. The control steers had a lower content of serum lipid initially than did either the safflower oil or

fat soy flour treated with 5% formaldehyde as one-third their diet for 1 week. In this study, the quantitative as well as qualitative composition of the serum lipid and individual serum lipoprotein fractions were studied in maturing beef steers consuming a high energy finishing ration which was supplemented with a formaldehyde treated protein and fat emulsion. A previous study employing steers receiving high energy diets supplemented with unprotected added fat demonstrated that serum lipid and lipoprotein fraction concentrations were quite stable regardless of the level of fat addition to the ration (Dryden et al., 1971). The current studies were conducted to determine if a dietary fat product, treated to be nonfermentable in the rumen, could cause a shift in the quality or quantity of the serum lipid or serum lipoprotein fractions of the bovine. Materials and Methods The rations containing no added fat, 6% added safflower oil or 12% added protected safflower oil and feeding periods utilized in this study were the same as those discussed by Cuitun et al. (1975). These authors have also discussed the information and composition of the formaldehyde treated casein:safflower oil (1:1, w/w) product in addition to in vitro studies on the stability of this protected safflower oil product. The steers were fed to approximately 454 kg and slaughtered. The fatty acid profile and lipid content of each of the rations as well as fatty acid composition of certain carcass tissues and 28-day subcutaneous fat biopsies have been determined by J. A. Marchello (unpublished data). Blood samples obtained by jugular vein puncture at 28-day intervals 4 hr after the 7:00 am feeding were allowed to clot in 50 ml centrifuge tubes for 24 hr at 4 C. The serum was separated by centrifugation at 24,000 X g for 10 min and EDTA-Na2 was added to 10-3M as an antioxidant and Merthiolate was added to 10-4 (w/v) as a preservative. The pH was adjusted to 7.4 and these conditions were, maintained throughout subsequent steps. Initially and at 84 and 168 days of the trial, large samples (200ml) of serum were collected for lipoprotein analysis. A Spiaco Model L preparative ultracentrifuge operating at 5 C was utilized to isolate by

4E. MerciaAG., Darmstadt, Germany.

PROTEIN PROTECTED FATS IN RUMINANTS the protected safflower oil supplemented animals which had very similar levels at about 340 rag/100 ml serum. This trend continued through the first 56 days of the experiment; however, during the next 28 days the serum lipid level of the steers consuming protected safflower oil demonstrated a marked increase to approximately 730 mg/100 ml serum. Although the elevation in serum lipid for the protected safflower oil diet was not statistically different ( P > .05) because of animal variation at 84 days, the additional increase demonstrated by these steers at 112 days caused their serum lipid level to be significantly greater (P < .05) than the control steers or the group consuming the unprotected safflower oil diet. The serum lipid level of the protected safflower oil steers continued to rise and be significantly higher (P < .05) than the other treatments throughout the remainder of the 168-day trial. On the final sample period, steers consuming the protected safflower oil had an average serum lipid level of 930 mg/100 ml serum which was 2.1 times greater than the control animals and 1.7 times greater than the animals supplemented with safflower oil. It was apparent that the protected safflower oil product which was not subjected to rumen fermentation but was hydrolyzed in the abomasum and then absorbed to a greater
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extent than the unprotected oil of the 6% safflower oil ration (Cuitun et al., 1975), caused a marked and nearly linear increase with time in total serum lipid. It should be noted that this rise in serum lipid occurred despite the fact that the concentrate level of the ration was maintained at 80% throughout the trial. A study of the literature generally indicates that serum lipid levels in the bovine decrease or remain relatively constant as the concentrate level of the ration is increased (Parham et al., 1950; Bohman et at., 1959, 1962; Marchello et al., 1971). These data suggest that dietary lipid supplements in the emulsified and formaldehyde treated form should enhance the efficiency of feed conversion by steers fed under feedlot conditions. However, general performance data from this trial (Cuitun et al., 1975) did not demonstrate an advantage to feeding the treated casein:safflower oil product. Feed efficiency in terms of feed per unit gain was not significantly different between the three diets. These data indicate that the bovine may not be able to efficiently metabolize larger quantities of fat which are typical of the diet of a monogastric animal. Previous studies with maturing steers have indicated that additions of 4 to 6% unprotected fat appears to be a practical maximum amount that can be supplemented to high concentrate rations containing 3 to 5% extractable lipid (Marchello etal., 1971, 1972; Figroid, 1971). Total serum lipid fatty acid profiles for steers on the three diets at 0, 56, 112 and 168 days into the trial revealed that the protected safflower oil supplemented diet was effective in preventing or reducing the extent of fatty acid hydrogenation in the rumen (table 1). At 56 days the level of serum linoleic acid increased significantly (P < .05) for steers consuming the protected safflower oil diet as compared to either of the other two rations. This effect was also present through the remaining two periods of the trial, however, the relative quantity of linoleic did not continue to increase for either of the safflower oil supplemented diets. There was a significant (P < .05) rise in the level of linoleic acid in the serum of the control animals for the last two periods of the trial as compared to the initial two periods. It has been previously reported that the quantity of serum iinoleic acid will increase (P < .05) with time for steers

700

DRYDEN ET AL. consuming a high energy ration which has not been supplemented with fat (Marchello et al., 1972, 1972). An inverse relationship between oleic acid and linoleic acid was noted, the former decreased while the latter increased (P > .05), for steers consuming the protected safflower oil diet. Furthermore, the relative percentage of the total unsaturated fatty acids in the serum between the three diets was not significantly different ( P < .05) at any one of the four sampling period. The two diets supplemented with safflower oil tended to have a greater percentage of total unsaturated fatty acids after the initial period. This result is surprising since the level of total serum lipid was more than doubled in the latter stages of the trial for the steers consuming the protected safflower oil diet compared to the other two diets (figure 1). One would expect the polyunsaturated fatty acids of the protected safflower oil diet to cause a shift in the percent unsaturated fatty acids in the serum for these animals. The serum LDL concentration for steers'on the three diets at three sampling periods during the trial is depicted in figure 2. The ration containing the protected safflower oil caused a significant ( P < .05) elevation in the serum LDL concentration of steers fed this diet 84 and 168 days as compared to the controls and the 6% safflower oil diet which did not differ ( P > .05). Such a marked increase (approximately fourfold over the control or safflower oil supplemented animals) or concentration (330 mg/lO0 ml serum) of LDL has not been previously reported in the literature for the bovine (Dryden et al., 1971; Wendlandt and Davis, 1973). The sharp rise in LDL concentration for the steers on the protected safflower oil diet paralleled the elevated total serum lipid levels observed for these same steers (figure 1). The serum HDL fraction was not as consistent in response to the dietary treatment as was the L D L fraction (figure 3). The level of serum HDL for the control animals was not different by period throughout the trial (P > .05). However, the serum HDL o f the steers supplemented with unprotected safflower oil was significantly (P < .05) lower at 84 and higher at 168 days than it was at the initiation of the trial. Why there was such a pronounced reduction in the level of HDL at 84 days in the trial for these steers is not apparent. Judging

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proximately 45% lipid) was greater (P < .05) on the final period for the protected safflower oil treatment then at the other two sampling periods, it was apparent that most of the elevation in total serum lipid noted for this treatment (figure 1) was due to an increase in the LDL (approximately 80% lipid) fraction. Table 2 contains the fatty acid profile of the major acids of the four lipid classes of the serum LDL and HDL lipid for periods and diets. Since large differences in fatty acid composition between the lipid classes existed, all statistical analyses were conducted on a within fraction basis with subsequent nesting of the main effects. The LDL glyceride and phospholipid lipid classes revealed the greatest response in fatty acid shift for the protected safflower oil diet as compared to the other two treatments. The level of linoleic acid in these two classes was in most cases elevated (P < .05) on both period 2 and 3 for this treatment. Furthermore, there was a decrease (P < .05) in oleic acid for the protected safflower oil treatment as compared to the control or the

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DRYDEN ET AL. lipoprotein fractions in this trial was very similar to data previously reported on this type of dietary supplementation (Dryden et al., 1971). The data presented in table 2 of this study demonstrated that the feeding of a protected safflower oil supplement to fattening steers does not alter the serum fatty acid profile to an appreciably greater extent than does the feeding of this highly unsaturated fat in the unprotected form at a similar level in the diet.

safflower oil supplemented steers. Although the quantities of total unsaturated fatty acids tended to be higher on the two diets to which safflower oil was supplemented, the differences were not significant (P > .05). The fatty acids of the free fatty acid and cholesterol ester lipid components of the LDL also demonstrated some response to the various treatments. However, with most acids the responses were not statistically significant (P > .05) shifts in fatty acid ratios. There was a trend toward elevated levels of C t 8 : 2 on the safflower oil supplemented diets but length of time on the diets did not appear to influence the extent of this elevation (table 2). When the level of C18:2 was elevated there was, in most cases, a decrease in quantity of C18:2 to asimilar extent which resulted in no change (P > .05) in the quantity of total unsaturated fatty acids between the three dietary treatments throughout the trial. The fatty acids of the cholesterol ester class did tend to have a greater percentage of total unsaturated fatty acids on the safflower oil supplemented diets but the protected safflower oil feeding did not cause a detectable response over the safflower oil ration towards enhanced levels of total unsaturated fatty acids. Unlike the fatty acids of the LDL phospholipids and glycerides, the fatty acids of the cholesterol esters or free fatty acid fractions in the HDL demonstrated essentially no differences (P > .05) between the three dietary treatments at the periods studied (table 2). There was a similar trend in these two classes for both the LDL and HDL fractions for C18:2 to be somewhat elevated on the safflower oil treatments and for C18:1 to be almost proportionately reduced. Changes in the percent total unsaturated fatty acids in these two lipid classes followed no consistent pattern. The HDL cholesterol ester fatty acids were altered by dietary treatment. Linoleic acid was significantly higher in this fraction for the steers consuming the protected safflower oil product at both periods 2 and 3 than it was for the control steers and oleic acid was reduced (P < .05) (table 2). The response of the steers on the unprotected safflower oil diet tended to be more like the steers fed the protected safflower oil than the controls. The influence of the unprotected safflower oil on the fatty acid composition of the four lipid classes o f both the L D L and the HDL

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PROTEIN PROTECTED FATS IN RUMINANTS Parham, A. P., R. W. Colby and J. K. Riggs. 1950. The influence of solvent extracted and hydraulic processed cottonseed meals upon performance and level of plasma carotene, vitamin A and fat in the blood of wintering beef cows. J. Anita. Sci. 9:560. Plowman, R. D., J. Bitman, C. H. Gordon, L. P. Dryden, H. K. Goering, L. F. Edmondson, R. A. Yoncoskie and F. W. Douglas, Jr., 1972. Milk fat

705

with increased polyunsaturated fatty acids. J. Dairy Sci. 55:204. Scott, T. W., P. J. Bready, A. J. Royal and L. J. Cook. 1972. Oil seed supplements for the production of polyunsaturated ruminant milk fat. Search 3:170. Wendlandt, R. M. and C. L. David. 1973. Characterization of bovine serum lipoproteins. J. Dairy Sei. 56:337.