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Lipid sources differ in caloric value for growing rats
NUTRITION RESEARCH, Vol. 12, pp. 407-418,1992 0271-5317/92 $5.00 + .00 Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
LIPID SOURCES DIFFER IN CALORIC VM~UE FOR GROWING RATS 1
D.A. Khalil, M.S., C.F. Hanson 2, Ph.D. and F.N. Owens3, Ph.D. Department of Nutritional Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A
ABSTRACT Growing male weanling rats (52 + 1.2 g) were fed a restricted basal diet (sucrose, protein, minerals, vitamins, corn oil) with addition of isocaloric amounts of sucrose or lipid (47 total keal/day). In the first experiment, diets contained added sucrose or beef tallow and either the required amount of calcium (62 mg/day) or a high level of calcium (230 mg/day). Substituting tallow for sucrose on a metabolizable energy (ME) basis increased (P<.0i) rate (4.8 vs 4.0 g/day) and efficiency of gain. Interactions between lipidlevel and added Ca were detected. Added Ca decreased digestibility of energy only with the tallow diet (98, 99, 95 and 80% for sucrose, sucrose plus Ca, tallow and tallow plus Ca, respectively). In experiment 2, a basal diet (18.5 keal/day) including 186 mg calcium/day was fed alone or with 25 kcal/day ME added from either sucrose, corn oil, tallow or coconut oil. Averaged across energy sources, addition of energy to the basal diet increased finalweight, dally gain and gain/feed ratio of the rats. Lipid, soap and energy content of feces were increased by added energy. Addedenergy decreased digestibility of energy, ether extract and total lipid (soap plus ether extract). Tallow was less w e l l digested than corn or coconut oil. Tallow-fed rats had less body lipids than rats fed corn oil but were similar in body composition to rats fed coconut oil. Net energy for gain, relative to sucrose at 1.00, was 3.35, 2.77 and 2.77 for corn oil, tallow and coconut oil, respectively. Physiological fuel values underestimated the net energy content of lipids compared to carbohydrate. The extra-caloric effect was greater for plant lipids than for tallow. Supplemental calcium depressed the digestibility and ME values of tallow. The higher value of corn oil than for tallow in these diets is due to decreased energy digestion and greater excretion of fecal soaps by rats fed tallow.
KEY WORDS: Tallow, Lipids, Energy, Calcium, Calories INTRODUCTION Caloric (physiological fuel) values for food components, derived by Atwater and Bryant in 1899, were estimates of the amount of energy avadable for metabolism (metabolizable energy) by animals and man (1). Digestible energy (DE) values were derived by multiplying the combustible energy content (gross energy) of each nutrient by its digestibility, For protein, but not for carbohydrate or lipid, an additional deduction from DE to calculate metabolizable energy (ME) was included for the energy excreted as urea. Net energy (NE) is ME minus heat increment (2). NE is used for maintenance and gain. NE for gain is 1 Journal Article No. 6033 of the Agr. Exp. Sta., Oklahoma State Univ., Stillwater, OK 74078. 2 Contact author. 3 Department of Animal Science. 407
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calculated from retention of consumed energy while NE for maintenance must be determined using animals neither gaining nor losing energy. When comparing the energy value of nutrients, some workers have used ME while others have used NE. In addii{on, some have used growing whereas others have used mature animals. The gross energy of food lipid that Atwater used was 9.47 kcal/g. Despite a slightly higher average energy value for animal than for vegetable triglycerides (9.43 vs 9.37 kcal/g), recently determined values (3) were quite close to the original estimate. However, values for animal products ranged from 8.08 to 9.49 due in part to the lower energy content of phospholipids. Based on ether extraction of feces, the value accepted for digestibility of lipid was 95%. Ether extraction, although acceptable for tissue lipid measurement, fails to recover fecal soap; approximately 44 to 97% of the lipid in feces of swine and other animals exists as the soap (4, 5). Thus, ether extraction typically underestimates total lipid in feces by over 50%. This causes digestibility and, thereby, the ME value for lipid to be overestimated. To recover fecal soaps, acidification must precede extraction of lipid. In one comparison, acidification prior to extraction of lipid from feces decreased apparent digestibility of lipid from 78% to 49% (6). Source and type of lipid and other diet components also markedly altered the digestibility of lipid (7). Rate and extent of digestion was greater for unsaturated than for saturated fatty acids. Sklan et al. (8) reported that intestinal digestibility values for sheep exceeded 90% for unsaturated fatty acids but fell to 55 and 65% for stearic acid and tristearin. For humans, absorption values of 78, 97, and 100% were determined for labeled stearic, oleic and linoleic acids, respectively (9). Other diet ingredients can modify lipid digestibility. Addition of calcium to the diet (1.94 vs .86%) increased fecal lipid excretion b), over 30%, possibly through increasing the formation of insoluble soaps in the intestine (10). In addition to differences in digestion, extent of metabolism of absorbed fatty acids may differ. By 9 hours after consumption, oxidation was 14 and 3 fold greater for oleic and linoleic than for stearic acid (9). Because fatty acid digestibility and metabolism can be altered by a number of inherent and dietary factors, the caloric value of lipid should not be considered to be a single or a constant value. Donato and Hegsted (11), based on body composition analysis of rats, indicated that the caloric value of lipid (Sprytm), relative to sucrose at 4 kcal/g, should be 11.1, not 9 kcal/g. Forbes et al. (12) and Carew et al. (13), also comparing lipid to carbohydrate at an assumed 4 kcal/g, calculated that the physiological fuel value of hpidexceeded 10 kcal per gram. For calonc values to exceed gross energy (9.47 kcal/g) is a thermodynamically impossible feat; therefore, either associative effects must be present or such comparisons are based upon net energy, not ME. Nevertheless, these high values suggest that the ratio of retained t o M E mnst be higher for lipid than for carbohydrate (14). Although the caloric value of protein and carbohydrate has been shown to vary with source (15, 16), the caloric value o f different sources of lipid in diets containing various calcium levels has not been studied. Such values are needed to accurately estimate energy values of foods for animals and humans. Hence, our objectives were: 1. To examine the utilization (both digested and retained) of calories from lipid versus sucrose. 2. To determine the effect of calcium supplementation on lipid digestibility. 3. To compare digestibilities and energy retentions from sucrose or several sources of lipid in diets containing high amounts of calcium. 4. To determine the impact of calorie source on carcass composition of rats.
CALORIC VALUE OF LIPIDS FOR RATS
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METHODS AND MATERIALS We conducted two experiments using slightly different treatments. The analytical methods for both experiments were similar and will be discussed together. We received approval from the University Animal Use Committee prior to conducting these experiments. Experiment 1. Thirty, male weanling Sprague-Dawley rats, 52 + 1.2 g mean weight, were caged separately in suspended stainless steel cages in a temperature and humidity controlled room with a 12 hr-Hght, 12 hr-dark cycle. All animals had ad libitum access to water throughout the trial. During a one-week pre-experiment adaptation period, the rats were fed a basal diet. Six rats were sacrificed to determifie initial b o ~ composition. The remaining rats were assigned to four treatments (six per treatment). Diets contained either sucrose or beef tallow added to a fortified basal mixture, with or without added calcium carbonate in a 2 by 2 factorial design. The effects of energy source (sucrose versus tallow), of added Ca and the interaction between energy source and Ca intake on rate of weight gain and body composition were tested, Intake of each animal was restricted to 47 kcal ME/day (Table 1). Experimental diets were formulated by adding to the fortified basal diet, the test nutrients: either sucrose or beef tallow with or without 168 nag daily extra calcium from CaCO3 (Table 1). Thus, the diets provided either a normal amount (62 rag/day) or an elevated amount (230 rag/day) of calcium. Of the ME in these diets, either 6.5% (sucrose diets) or 67% (tallow diets) came from lipid. The basal mixture contained casein, corn oil, sucrose, vitamin mix (AIN-76A), mineral mix (AIN-76), CaCO3, and choline chloride (17) to meet nutrient needs. Because a restricted amount of food was fed, most rats consumed all their food each day. Feed refused was removed and weighed. Final nutrient intakes were calculated as amount fed minus amount refused. The feeding period lasted 21 days after which the animals were sacrificed by CO2 suffocation. Feeding and animal care methods were the same as for Experiment 1. Six animals were sacrificed for initial body composition and thirty rats were assigned to five treatments with six animals per treatment. Diets in this experiment consisted of the basal diet fed alone (18.5 kcai ME/day) or this basal diet with 25 kcal ME/day added from either sucrose, corn oil, coconut oil or tallow (Table 1). This design allowed us to test with an elevated level of calcium intake (187 mg total Ca/day) the effects both of added energy and of source of supplemental energy (sucrose versus lipid; plant versus animal Hpid; saturation of the plant lipid) on carcass composition and energy retention. Of the calculated ME in these diets, lipid provided either 18% (basal diet), 8% (sucrose diet) or 65% (high lipid diet). Analysis of samples. In both trials, animals jgained weight throughout the trials (Tables 2 and 3) and exhibited no adverse reactions to their diets. Rats were weighed at the beginning of each experiment and weekly thereafter. All fecal matter was collected during the f'mal 10 days of each experiment and was considered to be representative of the total 21 days. Feces were weighed, and dried (100t' C for 48 hours) to calculate total fecal output and nutrient digestibilities. Immediately after the rats were sacrificed, the large intestines were removed; contents were rimed out with tap water and the empty large intestine was placed back into the body cavity. This prevented undigested food residues from influencing body composition. Carcasses (without removal of any organs or bodyparts) were frozen until analyzed. Carcasses then were autoclaved and ground with a foodblender. A sample of the ground carcass was lyophilized (a minimum of four days) to determine moisture content; these dried samples were used for all other analyses. Nitrogen content of feces and carcasses was determined following the AOAC Kjeldahl method (18) utilizing the Tecator Kjeltech instruments for digestion and distillation. Dried samples were pelleted and gross energy content was determined by oxygen bomb calorimetry (19).
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All feed and fecal samples were extracted initially with anhydrous ether (18) to determine the ether soluble lipid content of samples. In experiment 2, the soap content of the ether extracted residue also was measured using a modification of the technique described by Dole (20) as outlined below. For extraction of soap, a solution 40:10:1 isopropanol, heptane and sulfuric acid (1 Normal) was used. This extraction solution contained sulfuric acid to decrease the pH and release the fatty acids from soaps rendering them soluble in heptane. Each sample, after ether extraction, was placed in a test tube, 10 ml of this extraction solution was added and the tubes were shaken gently overnight. Heptane (4 ml) and deionized water (6 mi) were added to each tube causing separation of the solution into two layers; lipid in the heptane layer was recovered and measured gravimetrically. Apparent digestibilities were calculated from intakes and fecal output. Energy retentions were calculated from gross energy content of carcasses. Energy gain during each study was calculated by subtracting the initial from the final energy content. Digestibilities of nutrients, metabolizable and net energy forgain values and tissue deposition derived from each supplemental ingredient (Experiment 2) were calculated by subtracting appropriate measurements from rats fed the basal diet from those of rats fedthe supplemented diets. Heat increment was calculated as metabolizable minus retained energy (NE). For experiment 1, our experimental model was a 2 by 2 factorial; effects of energy source, calcium level and the interaction of calcium source and calcium level were estimated the General Linear Models procedure of SAS (21). Whenever an interaction was detected <.05), means were comparedby Duncan's Multiple Range Test. For Experiment 2, orthogonal contrasts were used to compare treatments. These contrasts included the effects of added energy (supplemented versus basal diet), of source of supplemental energy (sucrose versus lipid), of lipid source (plant versus animal) and of saturation of the plant lipid (corn versus coconut oil). Statistical analyses were performed and contrasts were testednsing the General Linear Models procedure of SAS (21).
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RESULTS Exveriment 1. Substituting tallow for sucrose calories increased (P <.01) rate (4.8 vs 4.0 g/day) and efficiency of gain averaged across calcium levels (Table 2). The increased weight gain at an equal calculated ME intake indicates that use of ME for weight gain was more efficient from tallow than from sucrose calories. More feces higher in lip"ld and energy content were produced by rats fed the lipid-supplemented than those fed the sucrosesupplemented diets. Thus, the digestibility of dry matter and energy were lower for the rats fed the lipid-supplemented diets. Nevertheless, energy retention was greater for rats fed tallow, especially when expressed per unit of DE. The increase in carcass protein was greater for rats fed the tallow diet matching their faster weight gain, Added calcium decreased final weight, dally gain and gain/feed ratio. Fecal dry matter output was increased by supplemental calcium, indicating that added calcium depressed dry matter digestibility. However, ether extract and energy content of feces were depressed, indicating that mineral content of feces was increased by feeding the higher calcium diet. Retention of energy was depressed by added calcium, largely reflecting the lower final rat weishts. Gain of carcass protein and lipid also was lower when diets were supplemented with calcium. Several interactions between added calcium and energy source were detected. Calcium had a greater effect on fecal dry matter output and on dry matter and energy digestibility with the tallow than with the sucrose diet. Added Ca decreased digestibility of energy only with the tallow diet (80% energy digestibility for tallow plus calcium, versus 98, 99 and 95% for sucrose, sucrose plus Ca and tallow diets, respectively). Added calcium depressed carcass protein gain more with the sucrose than with the tallow diet, but it tended to depress carcass lipid gain more with the tallow than with the sucrose diet.
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Despite a decrease of 14% in energy and dry matter digestibility from adding Ca to the tallow diet, ether extract digestibility was reduced by only 1.3%. Presumably, this reflects incomplete extraction of soaps by ether, which would cause lipid digestibility to be overestimated. Exveriment 2. Averaged across energy sources, addition of energy to the basal diet increased final weight, daily gain and gain/feed ratio of the rats (Table 3). Fecal dry matter output was increased by added tallow and coconut oil. Lipid, soap and energy content of feces were increased by added energy. Although digestibility of dry matter was increased by added energy, digestibility of energy, ether extract and total lipid (soap plus ether extract) was decreased by added energy (Table 3). Carcass deposition of lipid and protein were greater when energy was added to the basal diet. Compared with supplemental ME from sucrose, adding ME from lipid resulted in greater final weight, faster weight gain and a greater gain/feed ratio. The latter reflects the higher caloric density of lipid, but the former results indicate that ME was used more efficiently from lipid than from sucrose. Fecal dry matter output as well as ether extract, soap, total lipid and energy content of feces were greater from rats fed the lipid diets than from those fed the sucrose diet (Table 3). Thus, digestibility for both dietary and added dry matter and energy was greater for sucrose than lipids. Values for digestibility of added dry matter and energy, being slightly greater than 100%, may reflect slight errors in measurement or, alternatively, added nutrients may have a synergistic effect with the components of the basal diet resulting in a positive associative effect. Carcass gain in protein and lipid were greater with added lipids than with sucrose (Table 4). Despite the higher content of medium chain length fatty acids in coconut oil, digestibility was not greater for coconut oil than for corn oil. However, differences between corn oil and coconut oil may be attributed to the degree of saturation. Fecal dry matter excretion was greater for rats fed the more saturated plant lipid and, although ether extract content of feces tended to be lower, the percentage of soap, total lipid and energy in feces was higher for the coconut than for the corn oil diet. These differences caused digestibility of dry matter and energy, both of the total diet and of the added lipid, to be higher from corn oil than from coconut oil. Compared to rats fed coconut oil, rats fed corn oil had greater lipid deposition but less protein deposition (Table 4). The difference in lipid deposition parallels the differences in energy retention mentioned above. Effects of tallow were compared to the average of corn and coconut oil. The fatty acid saturation of tallow is approximately intermediate to these two plant lipids. Fecal dry matter output was ~reater with tallow (Table 3). As a percentage of fecal dry matter, ether extract, soap, total lipid and energy all were greater with tallow than with plant lipids. This caused digestibilities of dry matter, energy, ether extract, total lipid, added dry matter and added energy all to be lower with tallow. Tallow-fed rats were leaner than rats fed corn oil but were similar in body composition to rats fed coconut oil (Table 4). ME, the proportion of added gross energy from sucrose or lipid consumed that was digested, was higher for sucrose than for lipid, higher for corn oil than for coconut oil and lower for tallow than for the mean of corn and coconut oil (Table 3). Net energy for gain, the proportion of added gross energy consumed that was retained, was higher for lit~ids than for sucrose and higher for corn oil than for tallow and coconut oil (Table 4). Heat increment per gram fed was greater for lipids than for sucrose but lower for tallow than for the mean of the plant oils (Table 4). When expressed as a percentage of NE, heat increment was greater for the sucrose diet than for the diets with lipid added. The proportion of ME which was retained (NE) was greater for lipids than for sucrose. If net (retained) energy (kcal/g added) from Table 4 is calculated as a ratio with sucrose as 1.00, values for corn oil, tallow and coconut oil are 3.35, 2.77 and 2.77, respectively, rather than Atwater's 2.25 estimate.
DISCUSSION In Experiment 1, we examined the effect of the addition of approximately 2% calcium to diets on the growth, digestion and body composition of animals fedtallow or sucrose-
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supplemented diets. Substitution of tallow for sucrose decreased energy and dry matter digestibilities but increased rate and efficiency of weight gain. Feeding an elevat8ed level of calcium decreased energy digestibility of the tallow diet but not the sucrose diet, presumably due to formation of indigestible soaps in the intestinal tract. Soaps, being polar, are not extracted by anhydrous ether (22-24). Although soaps are not present in sizable quantities in meat products (25) or in most foods (24), some 44 to 97% of the lipid in feces is in the form of soap. Fecal soap concentrations may be increased by feeding elevated amounts of divalent cations, particularly calcium. Presence of soap in feces will lead to an overestimation of lipid digestibility. This causes digestibility and, thereby, the physiological fuel value for lipid t o b e overestimated. However, despite a decreased digestibility, conversion of ME to NE must have been greater for tallow than for sucrose because growth and energy retention both were greater. Thus, despite an overestimation of ME caused by soap formation, the NE value of lipid compared to carbohydrate may be underestimated by ME due to a lower heat increment for lipid. However, despite an increased efficiency of utilization of gross energy and DE, lipid content was not significantly greater for tallow-fed than sucrose-fed rats. This finding differs from the suggestion that high lipid diets cause body lipid content to increase (26). In Experiment 2, we measured the digestible, metabolizable and net energy of several sources of lipid: corn oil, coconut oil and tallow and compared them to sucrose in diets with supplemental calcium. Digestibility of added lipid was lower than that for added sucrose. In particular, digestibility of the animal lipid (tallow) was lower than for the other lipid sources, primarily due to an increase in soap content of feces. Amongthe plant sources, the saturated lipid source (coconut oil) was less extensively digested than the polyunsaturated lipid source (corn oil) as others have reported previously (27, 28). Again, conversion of ME to NE was greater for lipids than for sucrose as evidenced by greater growth and energy retention. More lipid was deposited b), rats fed the corn oil diet than for rats fed any other diet. Within the lipid supplemented diets, lipid content tended to be higher when the lipid source was less saturated. Awad et al. (29) detected no differences in lipid deposition but Shimomura et al. (30) reported that certain body organs were fatter for rats fed tallow than for rats fed safflower oil. DE and ME (physiological fuel values) for the lipid sources were calculated from the results of this trial. Measured ME values for sucrose, corn oil, tallow or coconut oil were 3.91, 8.48, 6.50 and 7.70 kcal/g added dry matter. These values are 98, 94, 72 and 86% of standard caloric values and agree with findings of other researchers (8, 9, 27, 28). ME averaged 1.93 times higher (P<.01) for lipid than for sucrose. Physiological fuel values, stating that lipid has 2.25 times the value of carbohydrate, overestimated the relative ME content of lipid by 14%. ME also was 20% lower (P<.01) for tallow than for the mean of corn and coconut oil. Although ME values for lipid appear to have been overestimated by standard caloric values, relative NE values paralleledcaloric values more closely. This was due to compensation from the lower heat increment per gram of lipid thanper gram of carbohydrate. NE values were 0.75, 2.51, 2.08 and 2.08 kcal-/g of added dry matter for sucrose, corn oil, tallow and coconut oil. Relative to the NEvalue of sucrose, NE values for corn oil, tallow and coconut oil were greater (P<.01), being 3.35, 2.77 and 2.77 times the value of sucrose, respectively. This compares to a NE value of sprytm 2.70 times that of sucrose based on data presented by Donato and Hegsted (11). Whether similar ratios would be expected for animals not depositing lipid is not certain. A constant ratio of net energy for growth to net energy for maintenance generally has been assumed but not tested. NE was 17% lower for tallow and for coconut oil than for corn oil. The calculated heat increment in kcal/g was higher for lipids than for sucrose and lowest for tallow among the lipids, perhaps due to less need for metabolism of fatty acids from tallow due to its similarity to depot fatty acids. When expressed relative to NE, heat increment was almost twice as great for sucrose than for lipid due to the need to convert carbohydrate to lipid for storage. The efficiency of conversion of ME to NE was higher for lipids than for sucrose but did not differ among lipid sources. The standard physiological fuel value for lipid underestimated the
CALORIC VALUE OF LIPIDS FOR RATS
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relative NE of corn oil by 49% and the relative NE for tallow and coconut oil by 23%. This indicates that lipid sources differ in their caloric value for growing animals. A similar difference between lipid sources might not be expected in NE for maintenance because at maintenance, most nutrients would be catabolized rather than converted into lipid for storage. In summary, physiological fuel values underestimated the net energy content of lipids compared to carbohydrate. Moreover, the extra-caloric effect was greater for plant lipids than for tallow. Supplemental calcium further depressed the DE and ME values of tallow. The higher value of corn oil than for tallow in these diets is due to decreased energy digestion and greater excretion of fecal soaps by rats fed tallow. The caloric value (ME) and the NE of corn oil were higher than for tallow and coconut oil, possibly due to level of saturation of fatty acids. Body composition was changed by altering the source of lipid in the diet. Substituting corn oil for tallow or coconut oil increased lipid and decreased protein deposition by rats. A more thorough understanding of how diet can alter body composition independent of body weight may prove valuable in both human and animal nutrition. Energy digestibility and retention were reduced by adding calcium to a high tallow diet. Whether c~cium depresses energy value of other lipid sources has not been investigated thoroughly. This effect may be important when considering calcium and lipid utilization by humans taking calcium supplements. Additionally, high lipid diets may reduce calcium availability from standard calcium supplements employed in animal and human diets. REFERENCES 1. Maynard LA. The Atwater system of calculating the caloric value of diets. J Nutr 1944;28:443-452. 2. National Research Council. Nutritional Energetics of Domestic Animals. Subcommittee on Biological Energy, Committee on Animal Nutrition, National Research Council, Washington, D.C.:National Academy Press 1981:4-27.
3. Miles C, Hardison N, Weihrauch JL, Prather E, Berlin E, Bodwell CE. Heats of combustion of chemically different lipids. J Am Dietet Assoc 1984; 84:659-664. 4. Harrison GA. Chemical examination of feces. In: Chemical methods in clinical medicine. London: J & A Churchill, Ltd., 1957:507-535. 5. Kahula S, Baumung A, Weissbach F. Determination of crude fat in feedstuffs and feces after treatment with hydrochloric acid. Arch Tierer 1983; 33:791-730. 6. Moore JA, Swindle RS, Hale WH. Effects of whole cottonseed, cottonseed oil or animal fat on digestibility of wheat straw diets by steers. J Anita Sci 1986; 63:1267-1273. 7. Ockner RK, Pittman JP, Yager JL. Differences in the intestinal absorption of saturated and unsaturated long chain fatty acids. Gastroent 1972; 62:981-992. 8. Sklan D, Arieli A, Chalupa W, Kronfeld DS. Digestion and absorption of lipids and bile salts in sheep fed stearic acid, oleic acid or tristearin. J Dairy So" 1985; 68:1667-1675. 9. Jones RTH, Pencharz PB, Clandinin Mr. Whole body oxidation of dietary fatty acids: Implications for energy utilization. Am J Clin Nutr 1985; 42:769-772. 10. Hines TG, Jacobson NL, Beitz DC, Littledike ET. Dietary calcium and vitamin D: risk factors in the development of atherosclerosis in young goats. J Nutr 1985; 115:167-178. 11. Donato K, Hegsted DM: Efficiency of utilization of various sources of energy for growth. Proc Natl Acad Sci 1985; 82:4866-4870. 12. Forbes EB, Swift RW, James WH, Bratzler JW, Black A. Further experiments on the relation of fat to economy of food utilization. J Nutr 1946; 32: 387-396.
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13. Carew LB, Hill FW. Effect of corn oil on metabolic efficiency of energy utilization by chicks. J Nutr 1964; 83: 293-299. 14. Anonymous. Role of fat and fatty adds in modulation of energy exchange. Nutr Rev 1988; 46:382-384. 15. Leveille GA, Cloutier PF. Isocalodc diets: Effects of dietary changes. Am J Clin Nutr 1987; 45:158-163. 16. Southgate ])AT, Durnin JVGA. Calorie conversion factors: An experimental reassessment of the factors used in the calculation of the energy value of human diets. Brit J Nutr 1970; 24:517-535. 17. American Institute of Nutrition: Ad Hoc Committee on Standards for Nutritional Studies. Report of the Committee. J Nutr 1977; 107: 1340. 18. Williams S ed. Official Methods of Analysis. 14th Ed. Arlington, VA. Association of Official Analytical Chemists, 1984. 19. Parr, 1261 Calorimeter. Parr Inst. Co. Moline, IL, 1988. 20. Dole VP. A relation between non-esterified fatty adds in plasma and the metabolism of glucose. J Clin Invest 1956:150-154. 21. SAS, Inst. Inc., Cary NC, 1985. 22. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; 37:911-917. 23. Heath TJ, Hill LN. Dietary and endogenous long-chain fatty acids in the intestine of sheep, with an appendix on their estimation in feeds, bile and feces. Aust J Biol Sci 1969; 22:1015-1029. 24. Hubbard WD, Sheppard A J, Newkirk DP, Prosser AR, Osgood T. Comparison of various methods for the extraction of total lipids, fatty adds, cholesterol, and other sterols from food products. J Am Oil Chem Soc 1977; 54:81-83. 25. Hagan SN, Murphy EW, Shelley LM. Extraction of lipids from raw beef lean using various solvent systems. J AOAC 1967; 50:250-255. 26. Schutz Y, Flatt JP, Jeuier E. Failure of dietary fat intake to promote fat oxidation: a factor favoring the development of obesity. Am J Clin Nutr 1989; 50:307-14. 27. Braude R, Newport MJ. Artificial rearing of pigs. Br J Nutr 1973; 29:447-455. 28. Cera KR, Mahan DC, Reinhart GA. Apparent fat digestibilities and performance responses of post weaning swine fed diets supplemented with coconut oil, corn oil or tallow. J Atom Sci 1989; 67:2040-2047. 29. Awad AB, Bernardis LL, Fink CS. Failure to demonstrate an effect of dietary fatty acid composition on body weight, body composition and parameters of lipid metabolism in mature rats. J Nutr 1990; 120:1277-1282. 30. Shimomura Y, Tamura T, Suzuki M. Less body fat accumulation in rats fed a safflower oil diet than in rats fed a beef tallow diet. J Nutr 1990; 120:1291-1296. Accepted for publication January 8, 1992.
LIPID SOURCES DIFFER IN CALORIC VM~UE FOR GROWING RATS 1
D.A. Khalil, M.S., C.F. Hanson 2, Ph.D. and F.N. Owens3, Ph.D. Department of Nutritional Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A
ABSTRACT Growing male weanling rats (52 + 1.2 g) were fed a restricted basal diet (sucrose, protein, minerals, vitamins, corn oil) with addition of isocaloric amounts of sucrose or lipid (47 total keal/day). In the first experiment, diets contained added sucrose or beef tallow and either the required amount of calcium (62 mg/day) or a high level of calcium (230 mg/day). Substituting tallow for sucrose on a metabolizable energy (ME) basis increased (P<.0i) rate (4.8 vs 4.0 g/day) and efficiency of gain. Interactions between lipidlevel and added Ca were detected. Added Ca decreased digestibility of energy only with the tallow diet (98, 99, 95 and 80% for sucrose, sucrose plus Ca, tallow and tallow plus Ca, respectively). In experiment 2, a basal diet (18.5 keal/day) including 186 mg calcium/day was fed alone or with 25 kcal/day ME added from either sucrose, corn oil, tallow or coconut oil. Averaged across energy sources, addition of energy to the basal diet increased finalweight, dally gain and gain/feed ratio of the rats. Lipid, soap and energy content of feces were increased by added energy. Addedenergy decreased digestibility of energy, ether extract and total lipid (soap plus ether extract). Tallow was less w e l l digested than corn or coconut oil. Tallow-fed rats had less body lipids than rats fed corn oil but were similar in body composition to rats fed coconut oil. Net energy for gain, relative to sucrose at 1.00, was 3.35, 2.77 and 2.77 for corn oil, tallow and coconut oil, respectively. Physiological fuel values underestimated the net energy content of lipids compared to carbohydrate. The extra-caloric effect was greater for plant lipids than for tallow. Supplemental calcium depressed the digestibility and ME values of tallow. The higher value of corn oil than for tallow in these diets is due to decreased energy digestion and greater excretion of fecal soaps by rats fed tallow.
KEY WORDS: Tallow, Lipids, Energy, Calcium, Calories INTRODUCTION Caloric (physiological fuel) values for food components, derived by Atwater and Bryant in 1899, were estimates of the amount of energy avadable for metabolism (metabolizable energy) by animals and man (1). Digestible energy (DE) values were derived by multiplying the combustible energy content (gross energy) of each nutrient by its digestibility, For protein, but not for carbohydrate or lipid, an additional deduction from DE to calculate metabolizable energy (ME) was included for the energy excreted as urea. Net energy (NE) is ME minus heat increment (2). NE is used for maintenance and gain. NE for gain is 1 Journal Article No. 6033 of the Agr. Exp. Sta., Oklahoma State Univ., Stillwater, OK 74078. 2 Contact author. 3 Department of Animal Science. 407
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calculated from retention of consumed energy while NE for maintenance must be determined using animals neither gaining nor losing energy. When comparing the energy value of nutrients, some workers have used ME while others have used NE. In addii{on, some have used growing whereas others have used mature animals. The gross energy of food lipid that Atwater used was 9.47 kcal/g. Despite a slightly higher average energy value for animal than for vegetable triglycerides (9.43 vs 9.37 kcal/g), recently determined values (3) were quite close to the original estimate. However, values for animal products ranged from 8.08 to 9.49 due in part to the lower energy content of phospholipids. Based on ether extraction of feces, the value accepted for digestibility of lipid was 95%. Ether extraction, although acceptable for tissue lipid measurement, fails to recover fecal soap; approximately 44 to 97% of the lipid in feces of swine and other animals exists as the soap (4, 5). Thus, ether extraction typically underestimates total lipid in feces by over 50%. This causes digestibility and, thereby, the ME value for lipid to be overestimated. To recover fecal soaps, acidification must precede extraction of lipid. In one comparison, acidification prior to extraction of lipid from feces decreased apparent digestibility of lipid from 78% to 49% (6). Source and type of lipid and other diet components also markedly altered the digestibility of lipid (7). Rate and extent of digestion was greater for unsaturated than for saturated fatty acids. Sklan et al. (8) reported that intestinal digestibility values for sheep exceeded 90% for unsaturated fatty acids but fell to 55 and 65% for stearic acid and tristearin. For humans, absorption values of 78, 97, and 100% were determined for labeled stearic, oleic and linoleic acids, respectively (9). Other diet ingredients can modify lipid digestibility. Addition of calcium to the diet (1.94 vs .86%) increased fecal lipid excretion b), over 30%, possibly through increasing the formation of insoluble soaps in the intestine (10). In addition to differences in digestion, extent of metabolism of absorbed fatty acids may differ. By 9 hours after consumption, oxidation was 14 and 3 fold greater for oleic and linoleic than for stearic acid (9). Because fatty acid digestibility and metabolism can be altered by a number of inherent and dietary factors, the caloric value of lipid should not be considered to be a single or a constant value. Donato and Hegsted (11), based on body composition analysis of rats, indicated that the caloric value of lipid (Sprytm), relative to sucrose at 4 kcal/g, should be 11.1, not 9 kcal/g. Forbes et al. (12) and Carew et al. (13), also comparing lipid to carbohydrate at an assumed 4 kcal/g, calculated that the physiological fuel value of hpidexceeded 10 kcal per gram. For calonc values to exceed gross energy (9.47 kcal/g) is a thermodynamically impossible feat; therefore, either associative effects must be present or such comparisons are based upon net energy, not ME. Nevertheless, these high values suggest that the ratio of retained t o M E mnst be higher for lipid than for carbohydrate (14). Although the caloric value of protein and carbohydrate has been shown to vary with source (15, 16), the caloric value o f different sources of lipid in diets containing various calcium levels has not been studied. Such values are needed to accurately estimate energy values of foods for animals and humans. Hence, our objectives were: 1. To examine the utilization (both digested and retained) of calories from lipid versus sucrose. 2. To determine the effect of calcium supplementation on lipid digestibility. 3. To compare digestibilities and energy retentions from sucrose or several sources of lipid in diets containing high amounts of calcium. 4. To determine the impact of calorie source on carcass composition of rats.
CALORIC VALUE OF LIPIDS FOR RATS
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METHODS AND MATERIALS We conducted two experiments using slightly different treatments. The analytical methods for both experiments were similar and will be discussed together. We received approval from the University Animal Use Committee prior to conducting these experiments. Experiment 1. Thirty, male weanling Sprague-Dawley rats, 52 + 1.2 g mean weight, were caged separately in suspended stainless steel cages in a temperature and humidity controlled room with a 12 hr-Hght, 12 hr-dark cycle. All animals had ad libitum access to water throughout the trial. During a one-week pre-experiment adaptation period, the rats were fed a basal diet. Six rats were sacrificed to determifie initial b o ~ composition. The remaining rats were assigned to four treatments (six per treatment). Diets contained either sucrose or beef tallow added to a fortified basal mixture, with or without added calcium carbonate in a 2 by 2 factorial design. The effects of energy source (sucrose versus tallow), of added Ca and the interaction between energy source and Ca intake on rate of weight gain and body composition were tested, Intake of each animal was restricted to 47 kcal ME/day (Table 1). Experimental diets were formulated by adding to the fortified basal diet, the test nutrients: either sucrose or beef tallow with or without 168 nag daily extra calcium from CaCO3 (Table 1). Thus, the diets provided either a normal amount (62 rag/day) or an elevated amount (230 rag/day) of calcium. Of the ME in these diets, either 6.5% (sucrose diets) or 67% (tallow diets) came from lipid. The basal mixture contained casein, corn oil, sucrose, vitamin mix (AIN-76A), mineral mix (AIN-76), CaCO3, and choline chloride (17) to meet nutrient needs. Because a restricted amount of food was fed, most rats consumed all their food each day. Feed refused was removed and weighed. Final nutrient intakes were calculated as amount fed minus amount refused. The feeding period lasted 21 days after which the animals were sacrificed by CO2 suffocation. Feeding and animal care methods were the same as for Experiment 1. Six animals were sacrificed for initial body composition and thirty rats were assigned to five treatments with six animals per treatment. Diets in this experiment consisted of the basal diet fed alone (18.5 kcai ME/day) or this basal diet with 25 kcal ME/day added from either sucrose, corn oil, coconut oil or tallow (Table 1). This design allowed us to test with an elevated level of calcium intake (187 mg total Ca/day) the effects both of added energy and of source of supplemental energy (sucrose versus lipid; plant versus animal Hpid; saturation of the plant lipid) on carcass composition and energy retention. Of the calculated ME in these diets, lipid provided either 18% (basal diet), 8% (sucrose diet) or 65% (high lipid diet). Analysis of samples. In both trials, animals jgained weight throughout the trials (Tables 2 and 3) and exhibited no adverse reactions to their diets. Rats were weighed at the beginning of each experiment and weekly thereafter. All fecal matter was collected during the f'mal 10 days of each experiment and was considered to be representative of the total 21 days. Feces were weighed, and dried (100t' C for 48 hours) to calculate total fecal output and nutrient digestibilities. Immediately after the rats were sacrificed, the large intestines were removed; contents were rimed out with tap water and the empty large intestine was placed back into the body cavity. This prevented undigested food residues from influencing body composition. Carcasses (without removal of any organs or bodyparts) were frozen until analyzed. Carcasses then were autoclaved and ground with a foodblender. A sample of the ground carcass was lyophilized (a minimum of four days) to determine moisture content; these dried samples were used for all other analyses. Nitrogen content of feces and carcasses was determined following the AOAC Kjeldahl method (18) utilizing the Tecator Kjeltech instruments for digestion and distillation. Dried samples were pelleted and gross energy content was determined by oxygen bomb calorimetry (19).
410
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All feed and fecal samples were extracted initially with anhydrous ether (18) to determine the ether soluble lipid content of samples. In experiment 2, the soap content of the ether extracted residue also was measured using a modification of the technique described by Dole (20) as outlined below. For extraction of soap, a solution 40:10:1 isopropanol, heptane and sulfuric acid (1 Normal) was used. This extraction solution contained sulfuric acid to decrease the pH and release the fatty acids from soaps rendering them soluble in heptane. Each sample, after ether extraction, was placed in a test tube, 10 ml of this extraction solution was added and the tubes were shaken gently overnight. Heptane (4 ml) and deionized water (6 mi) were added to each tube causing separation of the solution into two layers; lipid in the heptane layer was recovered and measured gravimetrically. Apparent digestibilities were calculated from intakes and fecal output. Energy retentions were calculated from gross energy content of carcasses. Energy gain during each study was calculated by subtracting the initial from the final energy content. Digestibilities of nutrients, metabolizable and net energy forgain values and tissue deposition derived from each supplemental ingredient (Experiment 2) were calculated by subtracting appropriate measurements from rats fed the basal diet from those of rats fedthe supplemented diets. Heat increment was calculated as metabolizable minus retained energy (NE). For experiment 1, our experimental model was a 2 by 2 factorial; effects of energy source, calcium level and the interaction of calcium source and calcium level were estimated the General Linear Models procedure of SAS (21). Whenever an interaction was detected <.05), means were comparedby Duncan's Multiple Range Test. For Experiment 2, orthogonal contrasts were used to compare treatments. These contrasts included the effects of added energy (supplemented versus basal diet), of source of supplemental energy (sucrose versus lipid), of lipid source (plant versus animal) and of saturation of the plant lipid (corn versus coconut oil). Statistical analyses were performed and contrasts were testednsing the General Linear Models procedure of SAS (21).
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RESULTS Exveriment 1. Substituting tallow for sucrose calories increased (P <.01) rate (4.8 vs 4.0 g/day) and efficiency of gain averaged across calcium levels (Table 2). The increased weight gain at an equal calculated ME intake indicates that use of ME for weight gain was more efficient from tallow than from sucrose calories. More feces higher in lip"ld and energy content were produced by rats fed the lipid-supplemented than those fed the sucrosesupplemented diets. Thus, the digestibility of dry matter and energy were lower for the rats fed the lipid-supplemented diets. Nevertheless, energy retention was greater for rats fed tallow, especially when expressed per unit of DE. The increase in carcass protein was greater for rats fed the tallow diet matching their faster weight gain, Added calcium decreased final weight, dally gain and gain/feed ratio. Fecal dry matter output was increased by supplemental calcium, indicating that added calcium depressed dry matter digestibility. However, ether extract and energy content of feces were depressed, indicating that mineral content of feces was increased by feeding the higher calcium diet. Retention of energy was depressed by added calcium, largely reflecting the lower final rat weishts. Gain of carcass protein and lipid also was lower when diets were supplemented with calcium. Several interactions between added calcium and energy source were detected. Calcium had a greater effect on fecal dry matter output and on dry matter and energy digestibility with the tallow than with the sucrose diet. Added Ca decreased digestibility of energy only with the tallow diet (80% energy digestibility for tallow plus calcium, versus 98, 99 and 95% for sucrose, sucrose plus Ca and tallow diets, respectively). Added calcium depressed carcass protein gain more with the sucrose than with the tallow diet, but it tended to depress carcass lipid gain more with the tallow than with the sucrose diet.
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Despite a decrease of 14% in energy and dry matter digestibility from adding Ca to the tallow diet, ether extract digestibility was reduced by only 1.3%. Presumably, this reflects incomplete extraction of soaps by ether, which would cause lipid digestibility to be overestimated. Exveriment 2. Averaged across energy sources, addition of energy to the basal diet increased final weight, daily gain and gain/feed ratio of the rats (Table 3). Fecal dry matter output was increased by added tallow and coconut oil. Lipid, soap and energy content of feces were increased by added energy. Although digestibility of dry matter was increased by added energy, digestibility of energy, ether extract and total lipid (soap plus ether extract) was decreased by added energy (Table 3). Carcass deposition of lipid and protein were greater when energy was added to the basal diet. Compared with supplemental ME from sucrose, adding ME from lipid resulted in greater final weight, faster weight gain and a greater gain/feed ratio. The latter reflects the higher caloric density of lipid, but the former results indicate that ME was used more efficiently from lipid than from sucrose. Fecal dry matter output as well as ether extract, soap, total lipid and energy content of feces were greater from rats fed the lipid diets than from those fed the sucrose diet (Table 3). Thus, digestibility for both dietary and added dry matter and energy was greater for sucrose than lipids. Values for digestibility of added dry matter and energy, being slightly greater than 100%, may reflect slight errors in measurement or, alternatively, added nutrients may have a synergistic effect with the components of the basal diet resulting in a positive associative effect. Carcass gain in protein and lipid were greater with added lipids than with sucrose (Table 4). Despite the higher content of medium chain length fatty acids in coconut oil, digestibility was not greater for coconut oil than for corn oil. However, differences between corn oil and coconut oil may be attributed to the degree of saturation. Fecal dry matter excretion was greater for rats fed the more saturated plant lipid and, although ether extract content of feces tended to be lower, the percentage of soap, total lipid and energy in feces was higher for the coconut than for the corn oil diet. These differences caused digestibility of dry matter and energy, both of the total diet and of the added lipid, to be higher from corn oil than from coconut oil. Compared to rats fed coconut oil, rats fed corn oil had greater lipid deposition but less protein deposition (Table 4). The difference in lipid deposition parallels the differences in energy retention mentioned above. Effects of tallow were compared to the average of corn and coconut oil. The fatty acid saturation of tallow is approximately intermediate to these two plant lipids. Fecal dry matter output was ~reater with tallow (Table 3). As a percentage of fecal dry matter, ether extract, soap, total lipid and energy all were greater with tallow than with plant lipids. This caused digestibilities of dry matter, energy, ether extract, total lipid, added dry matter and added energy all to be lower with tallow. Tallow-fed rats were leaner than rats fed corn oil but were similar in body composition to rats fed coconut oil (Table 4). ME, the proportion of added gross energy from sucrose or lipid consumed that was digested, was higher for sucrose than for lipid, higher for corn oil than for coconut oil and lower for tallow than for the mean of corn and coconut oil (Table 3). Net energy for gain, the proportion of added gross energy consumed that was retained, was higher for lit~ids than for sucrose and higher for corn oil than for tallow and coconut oil (Table 4). Heat increment per gram fed was greater for lipids than for sucrose but lower for tallow than for the mean of the plant oils (Table 4). When expressed as a percentage of NE, heat increment was greater for the sucrose diet than for the diets with lipid added. The proportion of ME which was retained (NE) was greater for lipids than for sucrose. If net (retained) energy (kcal/g added) from Table 4 is calculated as a ratio with sucrose as 1.00, values for corn oil, tallow and coconut oil are 3.35, 2.77 and 2.77, respectively, rather than Atwater's 2.25 estimate.
DISCUSSION In Experiment 1, we examined the effect of the addition of approximately 2% calcium to diets on the growth, digestion and body composition of animals fedtallow or sucrose-
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supplemented diets. Substitution of tallow for sucrose decreased energy and dry matter digestibilities but increased rate and efficiency of weight gain. Feeding an elevat8ed level of calcium decreased energy digestibility of the tallow diet but not the sucrose diet, presumably due to formation of indigestible soaps in the intestinal tract. Soaps, being polar, are not extracted by anhydrous ether (22-24). Although soaps are not present in sizable quantities in meat products (25) or in most foods (24), some 44 to 97% of the lipid in feces is in the form of soap. Fecal soap concentrations may be increased by feeding elevated amounts of divalent cations, particularly calcium. Presence of soap in feces will lead to an overestimation of lipid digestibility. This causes digestibility and, thereby, the physiological fuel value for lipid t o b e overestimated. However, despite a decreased digestibility, conversion of ME to NE must have been greater for tallow than for sucrose because growth and energy retention both were greater. Thus, despite an overestimation of ME caused by soap formation, the NE value of lipid compared to carbohydrate may be underestimated by ME due to a lower heat increment for lipid. However, despite an increased efficiency of utilization of gross energy and DE, lipid content was not significantly greater for tallow-fed than sucrose-fed rats. This finding differs from the suggestion that high lipid diets cause body lipid content to increase (26). In Experiment 2, we measured the digestible, metabolizable and net energy of several sources of lipid: corn oil, coconut oil and tallow and compared them to sucrose in diets with supplemental calcium. Digestibility of added lipid was lower than that for added sucrose. In particular, digestibility of the animal lipid (tallow) was lower than for the other lipid sources, primarily due to an increase in soap content of feces. Amongthe plant sources, the saturated lipid source (coconut oil) was less extensively digested than the polyunsaturated lipid source (corn oil) as others have reported previously (27, 28). Again, conversion of ME to NE was greater for lipids than for sucrose as evidenced by greater growth and energy retention. More lipid was deposited b), rats fed the corn oil diet than for rats fed any other diet. Within the lipid supplemented diets, lipid content tended to be higher when the lipid source was less saturated. Awad et al. (29) detected no differences in lipid deposition but Shimomura et al. (30) reported that certain body organs were fatter for rats fed tallow than for rats fed safflower oil. DE and ME (physiological fuel values) for the lipid sources were calculated from the results of this trial. Measured ME values for sucrose, corn oil, tallow or coconut oil were 3.91, 8.48, 6.50 and 7.70 kcal/g added dry matter. These values are 98, 94, 72 and 86% of standard caloric values and agree with findings of other researchers (8, 9, 27, 28). ME averaged 1.93 times higher (P<.01) for lipid than for sucrose. Physiological fuel values, stating that lipid has 2.25 times the value of carbohydrate, overestimated the relative ME content of lipid by 14%. ME also was 20% lower (P<.01) for tallow than for the mean of corn and coconut oil. Although ME values for lipid appear to have been overestimated by standard caloric values, relative NE values paralleledcaloric values more closely. This was due to compensation from the lower heat increment per gram of lipid thanper gram of carbohydrate. NE values were 0.75, 2.51, 2.08 and 2.08 kcal-/g of added dry matter for sucrose, corn oil, tallow and coconut oil. Relative to the NEvalue of sucrose, NE values for corn oil, tallow and coconut oil were greater (P<.01), being 3.35, 2.77 and 2.77 times the value of sucrose, respectively. This compares to a NE value of sprytm 2.70 times that of sucrose based on data presented by Donato and Hegsted (11). Whether similar ratios would be expected for animals not depositing lipid is not certain. A constant ratio of net energy for growth to net energy for maintenance generally has been assumed but not tested. NE was 17% lower for tallow and for coconut oil than for corn oil. The calculated heat increment in kcal/g was higher for lipids than for sucrose and lowest for tallow among the lipids, perhaps due to less need for metabolism of fatty acids from tallow due to its similarity to depot fatty acids. When expressed relative to NE, heat increment was almost twice as great for sucrose than for lipid due to the need to convert carbohydrate to lipid for storage. The efficiency of conversion of ME to NE was higher for lipids than for sucrose but did not differ among lipid sources. The standard physiological fuel value for lipid underestimated the
CALORIC VALUE OF LIPIDS FOR RATS
417
relative NE of corn oil by 49% and the relative NE for tallow and coconut oil by 23%. This indicates that lipid sources differ in their caloric value for growing animals. A similar difference between lipid sources might not be expected in NE for maintenance because at maintenance, most nutrients would be catabolized rather than converted into lipid for storage. In summary, physiological fuel values underestimated the net energy content of lipids compared to carbohydrate. Moreover, the extra-caloric effect was greater for plant lipids than for tallow. Supplemental calcium further depressed the DE and ME values of tallow. The higher value of corn oil than for tallow in these diets is due to decreased energy digestion and greater excretion of fecal soaps by rats fed tallow. The caloric value (ME) and the NE of corn oil were higher than for tallow and coconut oil, possibly due to level of saturation of fatty acids. Body composition was changed by altering the source of lipid in the diet. Substituting corn oil for tallow or coconut oil increased lipid and decreased protein deposition by rats. A more thorough understanding of how diet can alter body composition independent of body weight may prove valuable in both human and animal nutrition. Energy digestibility and retention were reduced by adding calcium to a high tallow diet. Whether c~cium depresses energy value of other lipid sources has not been investigated thoroughly. This effect may be important when considering calcium and lipid utilization by humans taking calcium supplements. Additionally, high lipid diets may reduce calcium availability from standard calcium supplements employed in animal and human diets. REFERENCES 1. Maynard LA. The Atwater system of calculating the caloric value of diets. J Nutr 1944;28:443-452. 2. National Research Council. Nutritional Energetics of Domestic Animals. Subcommittee on Biological Energy, Committee on Animal Nutrition, National Research Council, Washington, D.C.:National Academy Press 1981:4-27.
3. Miles C, Hardison N, Weihrauch JL, Prather E, Berlin E, Bodwell CE. Heats of combustion of chemically different lipids. J Am Dietet Assoc 1984; 84:659-664. 4. Harrison GA. Chemical examination of feces. In: Chemical methods in clinical medicine. London: J & A Churchill, Ltd., 1957:507-535. 5. Kahula S, Baumung A, Weissbach F. Determination of crude fat in feedstuffs and feces after treatment with hydrochloric acid. Arch Tierer 1983; 33:791-730. 6. Moore JA, Swindle RS, Hale WH. Effects of whole cottonseed, cottonseed oil or animal fat on digestibility of wheat straw diets by steers. J Anita Sci 1986; 63:1267-1273. 7. Ockner RK, Pittman JP, Yager JL. Differences in the intestinal absorption of saturated and unsaturated long chain fatty acids. Gastroent 1972; 62:981-992. 8. Sklan D, Arieli A, Chalupa W, Kronfeld DS. Digestion and absorption of lipids and bile salts in sheep fed stearic acid, oleic acid or tristearin. J Dairy So" 1985; 68:1667-1675. 9. Jones RTH, Pencharz PB, Clandinin Mr. Whole body oxidation of dietary fatty acids: Implications for energy utilization. Am J Clin Nutr 1985; 42:769-772. 10. Hines TG, Jacobson NL, Beitz DC, Littledike ET. Dietary calcium and vitamin D: risk factors in the development of atherosclerosis in young goats. J Nutr 1985; 115:167-178. 11. Donato K, Hegsted DM: Efficiency of utilization of various sources of energy for growth. Proc Natl Acad Sci 1985; 82:4866-4870. 12. Forbes EB, Swift RW, James WH, Bratzler JW, Black A. Further experiments on the relation of fat to economy of food utilization. J Nutr 1946; 32: 387-396.
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13. Carew LB, Hill FW. Effect of corn oil on metabolic efficiency of energy utilization by chicks. J Nutr 1964; 83: 293-299. 14. Anonymous. Role of fat and fatty adds in modulation of energy exchange. Nutr Rev 1988; 46:382-384. 15. Leveille GA, Cloutier PF. Isocalodc diets: Effects of dietary changes. Am J Clin Nutr 1987; 45:158-163. 16. Southgate ])AT, Durnin JVGA. Calorie conversion factors: An experimental reassessment of the factors used in the calculation of the energy value of human diets. Brit J Nutr 1970; 24:517-535. 17. American Institute of Nutrition: Ad Hoc Committee on Standards for Nutritional Studies. Report of the Committee. J Nutr 1977; 107: 1340. 18. Williams S ed. Official Methods of Analysis. 14th Ed. Arlington, VA. Association of Official Analytical Chemists, 1984. 19. Parr, 1261 Calorimeter. Parr Inst. Co. Moline, IL, 1988. 20. Dole VP. A relation between non-esterified fatty adds in plasma and the metabolism of glucose. J Clin Invest 1956:150-154. 21. SAS, Inst. Inc., Cary NC, 1985. 22. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; 37:911-917. 23. Heath TJ, Hill LN. Dietary and endogenous long-chain fatty acids in the intestine of sheep, with an appendix on their estimation in feeds, bile and feces. Aust J Biol Sci 1969; 22:1015-1029. 24. Hubbard WD, Sheppard A J, Newkirk DP, Prosser AR, Osgood T. Comparison of various methods for the extraction of total lipids, fatty adds, cholesterol, and other sterols from food products. J Am Oil Chem Soc 1977; 54:81-83. 25. Hagan SN, Murphy EW, Shelley LM. Extraction of lipids from raw beef lean using various solvent systems. J AOAC 1967; 50:250-255. 26. Schutz Y, Flatt JP, Jeuier E. Failure of dietary fat intake to promote fat oxidation: a factor favoring the development of obesity. Am J Clin Nutr 1989; 50:307-14. 27. Braude R, Newport MJ. Artificial rearing of pigs. Br J Nutr 1973; 29:447-455. 28. Cera KR, Mahan DC, Reinhart GA. Apparent fat digestibilities and performance responses of post weaning swine fed diets supplemented with coconut oil, corn oil or tallow. J Atom Sci 1989; 67:2040-2047. 29. Awad AB, Bernardis LL, Fink CS. Failure to demonstrate an effect of dietary fatty acid composition on body weight, body composition and parameters of lipid metabolism in mature rats. J Nutr 1990; 120:1277-1282. 30. Shimomura Y, Tamura T, Suzuki M. Less body fat accumulation in rats fed a safflower oil diet than in rats fed a beef tallow diet. J Nutr 1990; 120:1291-1296. Accepted for publication January 8, 1992.