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Fatty Acid Composition of Total Lipids and Glycerophospholipids of New Zealand White Rabbit Lean Muscle Tissue
The Professional Animal Scientist 10:40-45
Fatty Acid Composition of Total Lipids and Glycerophospholipids of New Zealand White Rabbit Lean Muscle Tissue C. C. AKOH Department of Food Science and Technology Food Science Building The University of Georgia Athens, GA 30602 C. V. NWOSU, S. D. LUKEFAHR, and D. R. RAO Department of Food Science and Animal Industries Schoo! of Agriculture and Home Economics Alabama A&M University, P. O. Box 264 Normal, AL 35762
differences between the fatty acid composition of male and female glycerophosphocholine (GPC), and glycerolysophosphocholine (GLPC). The preThe fatty acid composition of New Zealand White dominant fatty acids in GPC were C16:0 (36.1 moll rabbit lean tissue total lipids and individual glycerophospholipids was determined. Total lipid 100 moI) an d C18:2 n-6 (302 mol/100 mol) • The C 18:2 n-6 • content was between 2.5 and 3.0 g/100 g and levels in male and female rabbit glycerophosphoinositol (GPI) were statistically different (P<.05), glycerophosholipids were 26 to 29%. Neutral lipids account for 63 to 65% of the total lipids. The major representing 10.8 and 5.8 mol/100 mol, respectively. fatty acids of the total lipids were C182 n-6 (34.4 moll The overall major fatty acids of GPI were C18:0 (37.1 100 mol), C18:1 n-9 (26.4 moll100 mol), and C 160 (24.1 moll100 mol) and C20:4 n-6 (25.2 mol/100 mol). mol/100 mol). Total monoenes and polyunsaturated Glycerophosphoserine (GPS) and sphingomyelin fatty acids (PUFA) were 27.7 moll100 mol and 38.7 (SPH) had the highest overall concentrations of mol/100 mol, respectively. For monoenes and C18:0 , i.e., 41.0 and 44.2 mol/100 mol, respectively. PUFA, there were no statistically significant differSphingomyelin contained the highest overall conences1-P.>·OSJDetween male and female ralIDtt.... s·- -c-'-entratlon- oT total saturated fatty acids (83.3 moll However, there was marked heterogeneity in the 100 mol) followed by GLPC (60.1 mol/100 mol), relative fatty acid composition of the whereas the highest total monoenes and total PUFA were found in the GPE when compared with glycerophospholipids. In some cases there were significant differences (P<.05) between males and other glycerophospholipids. females with respect to individual fatty acids. High (Key Words: Total Lipids, Glycerophospholipids, concentrations of C 18:1 n-9 (26.8 moll100 mol) and C20:4 Rabbit, Lean Muscle Tissue, Fatty Acids.) n-6 (20.3 moll1oo mol) were found in glycerophosphoethanolamine (GPE). There were Introduction significant differences (P<.05) in C 16:0, C 18:2 n-6' and PUFA concentrations between the male and feThe rabbit has a great ability to proliferate and may become a good source of meat because of its male rabbits in GPE. There were no significant lower content of cholesterol (12, 13), total fat, and sodium compared with beef, chicken, and pork (13, 15). The rabbit meat industry is highly developed in Sponsored by D_W_ Weber. countries such as Russia, France, New Zealand, Reviewed by L_ J_ Bush and R Wander. Abstract
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LIPIDS OF RABBIT LEAN MUSCLE TISSUE
Italy, Spain, and some African countries like Ghana, but there is little production in the United States. The percentage of polyunsaturated fatty acids (PUFA) is high in rabbits, especially the essential fatty acid, linoleic acid (C'8:2n-s), according to previous reports (3, 4, 11, 13). In addition, rabbit meat has a lower concentration of stearic acid (C'8:0) and higher concentration of oleic acid (C'8:' n-9) compared with other animals (11, 13). Recent studies have suggested that diets high in oleic acid and PUFA, especially n-3 PUFA, may help patients with cardiovascular diseases (6, 8). Indeed, traditional Eskimos were said to consume diets high in monoenes and n-3 PUFA and low in C'8:2 n-S and arachidonic acid (C20:4 nos), which protected them against cardiovascular diseases. Akoh and Hearnsberger (2) recently demonstrated that diets high in C'8:' n-9 and n-3 PUFA have comparable effects in altering platelet lipid composition and blood-clotting factors, and may reduce the incidence of thrombosis. Rabbits are terrestrial animals and, thus, have low levels of long-chain n-3 PUFA. The health benefit of rabbit meat consumption may come from the moderate levels of monoenes found in the muscle tissue. Considerable amounts of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) are known to be present in most animal tissues (10). The fatty acid composition of rabbit total lipids, GPC, and GPE were recently reported (3, 4). To date, no report involving rabbit lean muscle tissue has been published on the composition of other glycerophospholipids such as glycerophosphoinositol (GPI), glycerophosphoserine (GPS), glycerolysophosphocholine (GLPC), and sphingomyelin (SPH). The objectives of the present work, therefore, were to determine the fatty acid composition of the total lipids and various glycerophospholipids of rabbit lean muscle tissue. Materials and Methods
Materials. Fatty acid methyl ester (FAME) standards and heptadecanoic acid were from NuChek Prep (Elysian, MN). Silica gel 60 plates were from M. Merck (Darmstadt, Germany). 8-Anilino-1-napthalenesulfonic acid ammonium salt (ANS) was from Aldrich Chemical Co. (Milwaukee, WI). All solvents were of HPLC grade.
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Methods. Rabbits of the New Zealand White breed (10 to 11 wk old), maintained at the International Small Livestock Research Center of Alabama A&M University were used. Four litters with two rabbits per litter (eight animals total) sampled at random, representing four males and four females, were involved in this study. Average body weights of male and female rabbits were 1766 and 1614 g, respectively. The rabbits were humanely slaughtered at 70 to 75 d of age, bled, skinned, and the muscle tissue carefully separated from the bones surgically with a knife. The subcutaneous fat was removed and the lean muscle tissue portion of the whole carcass was then minced in a blender and quickly frozen at -70·C until analysis (usually within 24 h). The lean muscle tissue was selected in place of a specific muscle because of the consumer demand for lean meats. Also the consumer is more likely to buy the whole carcass compared to individual muscle tissues. Lipids were extracted by the method of Folch et a!. (7). The chloroform-methanol used in the extraction contained .01 % butylated hydroxytoluene (BHT) to prevent oxidation. Total lipids were determined gravimetrically after drying in an air oven at 80·C. The neutral and individual glycerophospholipid classes were separated by TLC on silica gel 60 plates using chloroform:methanol:acetic acid:water, 50:37.5:3.5:2 vollvol/vol/vol as previously described, and weighed (1). Bands were detected under ultraviolet light after spraying with .1 % ANS. For fatty acid composition analysis, a known amount of heptadecanoic acid (C'7:0) as an internal standard was added to the total lipids or the isolated GPC, GPE, GPI, GPS, GLPC, and SPH classes prior to transesterification (5). Briefly, the scraped bands or total lipids were dissolved in 3 mL of anhydrous 6% HCI in methanol and incubated at 80·C for 2 h. For SPH, 3 mL of anhydrous 6% H2S04 in methanol was used for methyl ester preparation. After 2 h, the reaction mixture was cooled, and extracted with 2 mL hexane and 1 mL .1 M KC!. Hexane extraction was repeated. The hexane phase was passed through anhydrous sodium sulfate, and the FAME analyzed on a Hewlett Packard 5890A gas chromatograph equipped with a DB-225 30 meter fused silica capillary column (Ld ..25 mm, J&W Scientific, Folsom, CA). Helium carrier gas total flow was 20 ml/min, split ratio 1:66, and oven temperature
42
AKOH ET AL.
TABLE 1. Lipid composition of rabbit lean muscle meat". Lipid Total lipids Neutral Glycerophospholipids
Male
Female
gl100 9
gl100 9
2.5 63b 29
.,,, V.V
Overall SEM
x±
I")"'7...L
',I
..l.
~
.1
65 26
aValues are average of four rabbits each ± SEM. bpercentage of total lipids.
isothermally run -at 20S·C. The injector and detector temperatures were 2S0 and 260·C, respectively. We have found that under this isothermal condition iong chain faity acids of the n-3 series and C24:l n.g were separated within 30 to 34 min. Fatty acid identification was determined using retention times of knO\AJn standards. The data were analyzed by least squares analysis of variance procedures under a randomized block design. Litters were treated as the randomized block, and sex of rabbit (male vs female) was classified as a fixed main effect. Litter and residual random sources of variation were assumed to be normally and independently distributed with zero means and constant variances. Sex differences were tested for statistical significance using the F test from analysis of variance and at the P<.OS level of probability. All analyses were conducted using SAS® (14).
TABLE 2. Fatty acid composition (molesi100 mol) of rabbit lean muscle tissue total lipidsa. Fatty acid b
,... V14:0 C15:0 C16:0 C16:l n-7 C18:0 C18:l n·g C18:2n.6 C18:3n.6 C-18:3-n-3C20.0 C20:l n·g C20:2n.6 C20:3n·6 ,... V20:4n·6 Saturated Monoenes PUFA c
Overall x
Female
Male
2.1 .6 24.1 .9 6.8 26.4 34.4 .1
. 1
2.1 ± .1 .5 ± .1 24.1 ± .7 .9 ± .1 6.7 ± .7 26.5 ± .4 34.6 ± .7 .1 ± .0 2.4±J .1 ± .0 .3 ± .0 .2 ± .1 .1 ± .0 1.2 ± .2
2.0 .8 24.1 1.0 6.9 26.3 34.1 .1 2.2 .1 .3 .3 .2 1.5
33.7 ± .5 27.7 ± .4 38.7 ± .4
33.5 ± .6 27.7 ± .4 38.8 ± .4
33.9 ± .6 27.6 ± .4 38.5 ± .4
2.3 .1 .3 .2 .2 -4
,of
I .....
± .1 ± .1 ± .7 ± .1 ± .6 ± .4 ± .5 ± .0 ±-J ± .0 ± .0 ± .0 ± .0 ..J... ..!..
-t
± ± ± ± ± ± ± ± ± ± ± ± ±
.1 .1 .7 .1 .7 .4 .7 .0 .1 .0 .0 .1 .0 ± .2
aValues are molesi100 mol ± SEM from four whole carcasses per sex each. bOnly major fatty acids are reported. The values for and C22.6 n.3 fatty acids were less than .1%. cPUFA = polyunsaturated fatty acids.
C20:5n.3. C22:5n.6.
(34.4 mol/100 mol), C 18l n.9 (26.4 mol/100 mol), and C 16:0 (24.1 mol/100 mol). The level of C 180 (6.8 mol/ 100 mol) and C20:4 (1.4 mol/100 mol) were low. Monoenes and PUFA together contributed 66.4 mol/100 mol of the total fatty acids. This result differs significantly from the values reported by Cambero et al. (3), in which C 16:0 contributed 40% of the total lipids and C 18:2n.6' 11 %. According to CamResults and Discussion bero et al. (3), monoenes and PUFA together Table 1 shows the lipid composition of the rabbit represented 39.1% of the total fatty acids. Cambero - -lea-r+-mbisGI s-tissbls-.----T--Rs-t-GtaWif)iG-GGRt€ Rt-Gf- r-at>bit-et--al.-(dj--a_oo-~a_G-e-t-aI-.----(-lJ-)-Gl-iQ-RGt-~sf>Gft-aR-Y-meat was 2.S and 3.0 g/100 g for male and females, values for C20:2, C20:3, and C204 in the total lipids for respectively. The overall mean total lipid was 2.7 ± rabbits of similar age, apparently because they .7 g/100 g. Cambero et al. (3), Lukefahr et al. (12), used packed column GC in their analysis. and Rao et al. (13) have reported Slightly higher Our results were comparable to those reported values (4.9 to 7% total fat). Our results were most by Lee and Ahn (11) except that we detected more comparable to the 2.0 to 3.9% fat reported by Holfatty acids such as C 15:0, C 16:l n.7, C 18:3n.6, C20:l n.g, C20:2 mes et al. (9) and Zegarska et al. (16). The proporn.6, and C20:3n .6 with a capillary column. The commertion of neutral lipids to glycerophospholipids were cial diet consumed by the rabbits contained S.S% 63 and 29% and 65 and 26% for male and female fat (data not shown), whereas Cambero et al. (3) rabbits, respectively. fed 2.S% fat. Rabbit meat had a lower content of Table 2 shows the fatty acid composition of the total saturated fatty acids, 33.7 mol/100 mol, when rabbit lean muscle total lipids. There were no sigcompared with beef (S1.S mol/100 mol), pork (42.9 nificant differences in fatty acid composition bemol/100 mol), and chicken (36.S mol/100 mol) muscle tissue according to Lee and Ahn (11). Surpristween the maie - and female rabbits. The overaii mean values indicated that lean muscle tissue of ingly, we did not observe the presence of signifithe total carcass contained predominantly C 18:2 n.6 cant levels of C20:5 n.3, C22:5 n.6, and C22 :6 n-3 in the total
LIPIDS OF RABBIT LEAN MUSCLE TISSUE lipids. Our result is consistent with those reported by Cambero et al. (3, 4), Lee and Ahn (11), and Rao et al. (13). It may well be that the levels of these PUFA were too low to be detected under the conditions of our analysis. Table 3 shows the overall fatty acid composition of the glycerophospholipids (GPE, GPC, GPI, GLPC, and SPH). The predominant fatty acids in combined male and female rabbits GPE were C,s:, n-9 (26.8 mol/100 mol), C'S:2 n- S (24.2 moV100 mol), and C20:4 n-S (20.3 moV100 mOl). Notably, the level of PUFA was high and significant (P<.05) when the female (48.5 moV100 mol) was compared to male (42.9 moV100 mol) (data not shown). The amount of C'so and C'S:2 n-S was also significantly different between males and females. The females contained 30.1 moV100 mol C'S:2 n-S compared with 18.3 mol/100 mol in the males. Cambero et al. (4) also observed that females contain a higher concentration of C,s:2n-s than males. In general, the total saturated fatty acids were higher in male than in female rabbits. For GPC there were no significant differences between male and female rabbits in fatty acid composition. The overall composition indicated that the predominant fatty acids were C's:o (36.1 moV100 mol), C'S:2 n-S (30.2 moV100 mol), and C,s:, n-9 (18.7 moV100 mOl). The total saturated fatty acid content was higher than the monoenes and
43
PUFA. Cambero et al. (4) reported the fatty acid composition of GPE and GPC. They were unable to separate the other glycerophospholipid classes in the solvent system they used for TLC. No C20:4 noS' C20:5 n-3' and C22:S n-3 values were reported for GPC by Cambero et al. (4). Our results for GPC were in agreement with those reported by Cambero et al. (4) for C's:o and C,s:, n-9 but contrary to values reported for C's:o and C'S:2 noS' Cambero et al. (4) reported a value of 15 moV100 mol for C,s:2n-s, which represents half of the value reported here (30.2 moV100 mol). The values of C20:4 n-S (3.5 moll 100 mol) and C'S:3 n-3 (trace) that they reported for GPE were significantly lower than the values of 20.3 and 1.3 mo1/100 mol, respectively, reported here. In fact, Cambero et al. (4) reported that with the exception of very small amounts of C20:" which were always less than .7 mol/100 mol, they could not detect unsaturated fatty acids longer than C'B in glycerophospholipids. The relative proportions of individual glycerophospholipids classes as calculated after GLC in the presence of an internal standard were: GPC (55.4%), GPE (22.2%), GPI (11.0%), GPS (5.3%), SPH (4.4%), and GLPC (1.7%). Cambero et al. (4) and Kuksis (10) have reported similar values of 40 to 66% for GPC and 17 to 22% for GPE. The level of C'B:2 n-S in GPI was significantly different P<.05 in males (10.8 moV100
TABLE 3. Composition (molesl100 mol) of glycerophospholipids of rabbit lean muscle tissue ab . Fatty acid c GPS GPE GPC GPI 1.5 ± .1 1.1 ± .4 C'4:0 .6 ± .1 C'5:0 17.7 ± 1.3 12.3 ± 1.5 36.1 ± .8 13.2 ± 1.1 C,s:o .8 ± .1 C,s:, n.7 12.6 ± 1.8 7.7±1.1 37.1 ± 2.6 41.0 ± 3.6 C,s:o 23.7 ± 2.3 26.8 ± 1.6 18.7 ± .6 16.3 ± 1.6 C,s:, n.9 16.2 ± 1.5 24.2 ± 2.6 30.2 ± .8 8.3 ± .7 C'S:2 n·S 1.3 ± .3 C'S:3n.3 C20:0 20.3 ± 2.9 25.2 ± 2.0 Trd 7.2 ± .8 C20:4 n-S C22:0 C24:0 C24:' n-9 Saturated 26.6 ± 1.4 43.8 ± 1.8 50.2 ± 1.7 60.2 ± 2.2 23.7 ± 2.3 Monoenes 27.6 ± 1.6 18.7 ± .6 16.3 ± 1.6 PUFA 16.2 ± 1.5 45.7 ± 1.2 37.4 ± 1.4 33.6 ± 2.0
GLPC 5.6 ± .6
SPH 2.7 ±
39.1 ± 1.0
26.7 ± 1.3
15.4 ± 1.5 27.4 ± 1.7 12.5 ± 1.0
44.2 ± 1.2 9.3 ± .9 4.8 ± .5 4.9 ±
60.1 ± 2.2 27.4 ± 1.7 12.5 ± 1.0
.1
.5
2.4 ± .2 2.2 ± .2 2.6 ± .3 83.3 ± 1.3 11.9 ± 1.1 4.8 ± .4
aValues are molesl100 mol ±SEM from four male and four female carcasses. bGPE =glycerophosphoethanolamine; GPC =glycerophosphocholine; GPI =glycerophosphoinositol; GPLC =glycerolysophosphocholine; SPH = sphingomyelin; and PUFA =polyunsaturated fatty acid. COnly major fatty acids are reported. drr = trace amounts, less than .1%. Values for C20:5n-3, C22:5n-s, and C22:Sn-3were also less than .1%.
AKOH ET AL.
44
TABLE 4. Variancecomponent analysis of rabbit lean muscle tissue fatty mol) vs females (5.8 mo1/100 mol). The predomiacid for among-litter and within-litter (residual) sourcesab . nant fatty acids in GPI were, as expected, C 180 (37.1 Variance component (%) moV100 mol), C20: 4 n. 6 (25.2 moV100 mol), C18:1 n.9 ':I ..... ,,1/100 ..... ,,1\ Clnrl r 11':1" "'1"'11/1 ()() Fatty acids Residual Litters \'1 c:: v.v v v16:0 \ vv ..,,,,1\ Total lipid Cambero et al. (4) were unable to detect GPI in (92.5) (7.5) 2.171 .177 C16:0 their preparations. (85.4) 1.581 .271 (14.6) C18:0 Except for C14:0, there were no significant differ(94.1) (5.9) .080 .005 C18:3n.3 (14.9) PUFA .640 (85.1 ) .112 ences in the fatty acid composition in male and GPE female rabbit GPS (Table 3). Stearic acid C 18:0 (41.0 (86.6) .098 (13.4) .638 C14:0 9;063 (89.9) 1.021 (11.1 ) C16:0 mol/1OO molT was the preaominant fatty acid. Sur(91.3) (8.7) .035 .003 C16:1 n -7 prisingly, only traces of C20:4 n.6 were observed in (83.2) (16.8) .579 .117 C18:3n-3 GPS. No significant differences were observed in aOnlyfatty acids significantly influenced (P<.05) by the litter source of fatty acid composition bett/Jsen male and female variation are pi6santad in the table. bGPE = glycerophosphoethanolamine; PUFA =polyunsaturated fatty rabbit GLPC. The major fatty acids were C 16:0 (39.1 acid. moV100 mol) and C18:1n.9 (27.4 mol/100 mol). Notably, there was no C20: 4 n.5 in the GLPC, indicating that the GLPC was derived from possible hydrolysis by phospholipase A2 at the Sn-2 position. Totion for total lipids percentage has been previously gether, C16:0 and C18:0 contributed approximately 71.0 reported by Lukefahr et al. (12). Further research moV100 mol of the total fatty acids in the rabbit involving larger samples of rabbits would be useful SPH. Saturated fatty acids represented 83.3 moll to confirm present experimental results. This report 100 mol of the total fatty acids. As with other animal represents the first detailed analyses of the inSPH, the characteristic C20:0' C22:0, C24:0, and C24:1 n.9 dividual phospholipid class fatty acid composition of fatty acids were observed in rabbit meat and comrabbit lean muscle tissue. Domestic rabbit meat pare well to those reported by Kuksis (10). There consumption has not increased in recent years. Increasing of C18:1n.9 content of rabbit lean muscle were significant differences between male and female rabbits in content of C14:0, C 18:0, and total tissue may have potential health benefit in view of monoenes (data not shown). Of all the the fact that diets high in monoenes have been glycerophospholipids, SPH had the lowest amount suggested to have cholesterol-lowering effect and of PUFA and the highest amount of saturated fatty needs to be explored through breeding programs. Of particular concern is the fact that little or no long acids. There were some dimethylacetals formed during FAME preparation. However, their values chain n-3 PUFA were detected in both the total _are oat rep.o.ded io this....p.aFP=eL.cr_ _ _ _ _ _ _ _...;I~ipids and glycerophospholipid classes of this rabbit Overall, there was marked heterogeneity in the bree-d-:- -- ---- - fatty acid composition of the glycerophospholipid classes. Glycerophosphocholine and GPE were the Literature Cited major glycerophospholipid classes in rabbit lean 1. Akoh, C. C., and R. S. Chapkin. 1990. Composition of mouse muscle tissue, representing 55.4 and 22.2%, peritoneal macrophage phospholipid molecular species. Lipids 25: 613. respectively. It should be noted that (Table 4) there 2. Akoh, C. C., and J . O. Hearnsberger. 1991. Effect of catfish and was significant among-litter variation in some fatty salmon diet on platelet phospholipid and blood clotting in healthy men. J . Nutr. Biochem. 2:329. acids, inferring genetic and(or) common environ3. Cambero, M. I., L. de La Hoz, B. Sanz, and J. A. Ordonez. 1991. Lipid mental causes. The sex of the rabbits also conand fatty acid composition of rabbit meat: Part 1. - Apolar fraction. Meat Sci. 29:153. tributed in part to the total variance. Specifically, in 4. Cambero, M. I., L. de La Hoz, B. Sanz, and J. A. Ordonez. 1991. Lipid total lipids, only C16:0, C18:0, C 18:3n.3, and total PUFA and fatty acid composition of rabbit meat: Part 2. - Phospholipids. Meat Sci. 29:167. were significantly influenced by the litter source, 5. Chapkin, R. S., S. D. Somers, and K. L. Erickson. 1988. Dietary whereas in GPE, only C 14:0, C 16:0, and C 16:1n. 7 were manipulation of macrophage phospholipid classes: Selective increase in dihomogammalinolenic acid. Lipids 23:766. affected. The exact cause(s) explaining this source 6. Dyerberg, J ., H. O. Bang, E. Stofferson, S. Moncade, and J. R.Vane. of variation cannot be elucidated and thus warrant 1978. Eicosapentaenoic acid and prevention of thrombosis and further investigation. Significant among-litter variaatherosclerosis. Lancet ii:117. IIIVII
I
IIIVI"
UIIU
n."".~
111'-'11
I
111'VI}o
LIPIDS OF RABBIT LEAN MUSCLE TISSUE 7. Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. BioI. Chem. 226:497. 8. Grundy, S. M. 1986. Comparison of monounsaturated fatty acids and carbohydrates for lowering plasma cholesterol. New Engl. J. Med. 314:745. 9. Holmes, Z. A., S. F. Wei, D. J. Harris, P.R. Cheeke, and N. M. Patton. 1984. Proximate composition and sensory characteristics of meat from rabbits fed three levels of alfalfa meal. J. Anim. Sci. 58:62. 10. Kuksis, A. 1985. Animal lecithins. In: B. F. Szuhaj and G. R. List (Ed). Lecithins. p 105. American Oil Chemists Society, Champaign, IL. 11. Lee, Y. C., and H. S. Ahn. 19n. Studies on lipids and proteins of rabbit meat. 1. Emphasis on lipid components of rabbit meat. Korean J. Nutr. 10:78.
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12. Lukefahr, S. D., C. V. Nwosu, and D. R. Rao. 1989. Cholesterol level of rabbit meat and trait relationships among growth, carcass and lean yield performances. J. Anim. Sci. 67:2009. 13. Rao, D. R., C. B. Chawan, C. P. Chen, and G. R. Sunki. 1979. Nutritive value of rabbit meat. In: The Domestic Rabbit: Potentials, Problems and Current Research. p 53. Oregon State University Press, Corvallis, OR. 14. SAS. 1985. SAS User's Guide: Statistics (Version 5 Ed.). SAS Institute Inc., Cary, NC. 15. USDA. 1989. Composition of Foods, Raw, Processed, Prepared. Agriculture Handbook 8. ARS, USDA, Washington, DC. 16. Zegarska, Z., K. Markiewicz, and S. Smoczynski. 1979. Composition of fatty acids in muscle fat and depot fat of rabbits. Zesz. Nauk. Art. Olsz. Technol. Zywnosci. 15:167.
Fatty Acid Composition of Total Lipids and Glycerophospholipids of New Zealand White Rabbit Lean Muscle Tissue C. C. AKOH Department of Food Science and Technology Food Science Building The University of Georgia Athens, GA 30602 C. V. NWOSU, S. D. LUKEFAHR, and D. R. RAO Department of Food Science and Animal Industries Schoo! of Agriculture and Home Economics Alabama A&M University, P. O. Box 264 Normal, AL 35762
differences between the fatty acid composition of male and female glycerophosphocholine (GPC), and glycerolysophosphocholine (GLPC). The preThe fatty acid composition of New Zealand White dominant fatty acids in GPC were C16:0 (36.1 moll rabbit lean tissue total lipids and individual glycerophospholipids was determined. Total lipid 100 moI) an d C18:2 n-6 (302 mol/100 mol) • The C 18:2 n-6 • content was between 2.5 and 3.0 g/100 g and levels in male and female rabbit glycerophosphoinositol (GPI) were statistically different (P<.05), glycerophosholipids were 26 to 29%. Neutral lipids account for 63 to 65% of the total lipids. The major representing 10.8 and 5.8 mol/100 mol, respectively. fatty acids of the total lipids were C182 n-6 (34.4 moll The overall major fatty acids of GPI were C18:0 (37.1 100 mol), C18:1 n-9 (26.4 moll100 mol), and C 160 (24.1 moll100 mol) and C20:4 n-6 (25.2 mol/100 mol). mol/100 mol). Total monoenes and polyunsaturated Glycerophosphoserine (GPS) and sphingomyelin fatty acids (PUFA) were 27.7 moll100 mol and 38.7 (SPH) had the highest overall concentrations of mol/100 mol, respectively. For monoenes and C18:0 , i.e., 41.0 and 44.2 mol/100 mol, respectively. PUFA, there were no statistically significant differSphingomyelin contained the highest overall conences1-P.>·OSJDetween male and female ralIDtt.... s·- -c-'-entratlon- oT total saturated fatty acids (83.3 moll However, there was marked heterogeneity in the 100 mol) followed by GLPC (60.1 mol/100 mol), relative fatty acid composition of the whereas the highest total monoenes and total PUFA were found in the GPE when compared with glycerophospholipids. In some cases there were significant differences (P<.05) between males and other glycerophospholipids. females with respect to individual fatty acids. High (Key Words: Total Lipids, Glycerophospholipids, concentrations of C 18:1 n-9 (26.8 moll100 mol) and C20:4 Rabbit, Lean Muscle Tissue, Fatty Acids.) n-6 (20.3 moll1oo mol) were found in glycerophosphoethanolamine (GPE). There were Introduction significant differences (P<.05) in C 16:0, C 18:2 n-6' and PUFA concentrations between the male and feThe rabbit has a great ability to proliferate and may become a good source of meat because of its male rabbits in GPE. There were no significant lower content of cholesterol (12, 13), total fat, and sodium compared with beef, chicken, and pork (13, 15). The rabbit meat industry is highly developed in Sponsored by D_W_ Weber. countries such as Russia, France, New Zealand, Reviewed by L_ J_ Bush and R Wander. Abstract
40
LIPIDS OF RABBIT LEAN MUSCLE TISSUE
Italy, Spain, and some African countries like Ghana, but there is little production in the United States. The percentage of polyunsaturated fatty acids (PUFA) is high in rabbits, especially the essential fatty acid, linoleic acid (C'8:2n-s), according to previous reports (3, 4, 11, 13). In addition, rabbit meat has a lower concentration of stearic acid (C'8:0) and higher concentration of oleic acid (C'8:' n-9) compared with other animals (11, 13). Recent studies have suggested that diets high in oleic acid and PUFA, especially n-3 PUFA, may help patients with cardiovascular diseases (6, 8). Indeed, traditional Eskimos were said to consume diets high in monoenes and n-3 PUFA and low in C'8:2 n-S and arachidonic acid (C20:4 nos), which protected them against cardiovascular diseases. Akoh and Hearnsberger (2) recently demonstrated that diets high in C'8:' n-9 and n-3 PUFA have comparable effects in altering platelet lipid composition and blood-clotting factors, and may reduce the incidence of thrombosis. Rabbits are terrestrial animals and, thus, have low levels of long-chain n-3 PUFA. The health benefit of rabbit meat consumption may come from the moderate levels of monoenes found in the muscle tissue. Considerable amounts of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) are known to be present in most animal tissues (10). The fatty acid composition of rabbit total lipids, GPC, and GPE were recently reported (3, 4). To date, no report involving rabbit lean muscle tissue has been published on the composition of other glycerophospholipids such as glycerophosphoinositol (GPI), glycerophosphoserine (GPS), glycerolysophosphocholine (GLPC), and sphingomyelin (SPH). The objectives of the present work, therefore, were to determine the fatty acid composition of the total lipids and various glycerophospholipids of rabbit lean muscle tissue. Materials and Methods
Materials. Fatty acid methyl ester (FAME) standards and heptadecanoic acid were from NuChek Prep (Elysian, MN). Silica gel 60 plates were from M. Merck (Darmstadt, Germany). 8-Anilino-1-napthalenesulfonic acid ammonium salt (ANS) was from Aldrich Chemical Co. (Milwaukee, WI). All solvents were of HPLC grade.
41
Methods. Rabbits of the New Zealand White breed (10 to 11 wk old), maintained at the International Small Livestock Research Center of Alabama A&M University were used. Four litters with two rabbits per litter (eight animals total) sampled at random, representing four males and four females, were involved in this study. Average body weights of male and female rabbits were 1766 and 1614 g, respectively. The rabbits were humanely slaughtered at 70 to 75 d of age, bled, skinned, and the muscle tissue carefully separated from the bones surgically with a knife. The subcutaneous fat was removed and the lean muscle tissue portion of the whole carcass was then minced in a blender and quickly frozen at -70·C until analysis (usually within 24 h). The lean muscle tissue was selected in place of a specific muscle because of the consumer demand for lean meats. Also the consumer is more likely to buy the whole carcass compared to individual muscle tissues. Lipids were extracted by the method of Folch et a!. (7). The chloroform-methanol used in the extraction contained .01 % butylated hydroxytoluene (BHT) to prevent oxidation. Total lipids were determined gravimetrically after drying in an air oven at 80·C. The neutral and individual glycerophospholipid classes were separated by TLC on silica gel 60 plates using chloroform:methanol:acetic acid:water, 50:37.5:3.5:2 vollvol/vol/vol as previously described, and weighed (1). Bands were detected under ultraviolet light after spraying with .1 % ANS. For fatty acid composition analysis, a known amount of heptadecanoic acid (C'7:0) as an internal standard was added to the total lipids or the isolated GPC, GPE, GPI, GPS, GLPC, and SPH classes prior to transesterification (5). Briefly, the scraped bands or total lipids were dissolved in 3 mL of anhydrous 6% HCI in methanol and incubated at 80·C for 2 h. For SPH, 3 mL of anhydrous 6% H2S04 in methanol was used for methyl ester preparation. After 2 h, the reaction mixture was cooled, and extracted with 2 mL hexane and 1 mL .1 M KC!. Hexane extraction was repeated. The hexane phase was passed through anhydrous sodium sulfate, and the FAME analyzed on a Hewlett Packard 5890A gas chromatograph equipped with a DB-225 30 meter fused silica capillary column (Ld ..25 mm, J&W Scientific, Folsom, CA). Helium carrier gas total flow was 20 ml/min, split ratio 1:66, and oven temperature
42
AKOH ET AL.
TABLE 1. Lipid composition of rabbit lean muscle meat". Lipid Total lipids Neutral Glycerophospholipids
Male
Female
gl100 9
gl100 9
2.5 63b 29
.,,, V.V
Overall SEM
x±
I")"'7...L
',I
..l.
~
.1
65 26
aValues are average of four rabbits each ± SEM. bpercentage of total lipids.
isothermally run -at 20S·C. The injector and detector temperatures were 2S0 and 260·C, respectively. We have found that under this isothermal condition iong chain faity acids of the n-3 series and C24:l n.g were separated within 30 to 34 min. Fatty acid identification was determined using retention times of knO\AJn standards. The data were analyzed by least squares analysis of variance procedures under a randomized block design. Litters were treated as the randomized block, and sex of rabbit (male vs female) was classified as a fixed main effect. Litter and residual random sources of variation were assumed to be normally and independently distributed with zero means and constant variances. Sex differences were tested for statistical significance using the F test from analysis of variance and at the P<.OS level of probability. All analyses were conducted using SAS® (14).
TABLE 2. Fatty acid composition (molesi100 mol) of rabbit lean muscle tissue total lipidsa. Fatty acid b
,... V14:0 C15:0 C16:0 C16:l n-7 C18:0 C18:l n·g C18:2n.6 C18:3n.6 C-18:3-n-3C20.0 C20:l n·g C20:2n.6 C20:3n·6 ,... V20:4n·6 Saturated Monoenes PUFA c
Overall x
Female
Male
2.1 .6 24.1 .9 6.8 26.4 34.4 .1
. 1
2.1 ± .1 .5 ± .1 24.1 ± .7 .9 ± .1 6.7 ± .7 26.5 ± .4 34.6 ± .7 .1 ± .0 2.4±J .1 ± .0 .3 ± .0 .2 ± .1 .1 ± .0 1.2 ± .2
2.0 .8 24.1 1.0 6.9 26.3 34.1 .1 2.2 .1 .3 .3 .2 1.5
33.7 ± .5 27.7 ± .4 38.7 ± .4
33.5 ± .6 27.7 ± .4 38.8 ± .4
33.9 ± .6 27.6 ± .4 38.5 ± .4
2.3 .1 .3 .2 .2 -4
,of
I .....
± .1 ± .1 ± .7 ± .1 ± .6 ± .4 ± .5 ± .0 ±-J ± .0 ± .0 ± .0 ± .0 ..J... ..!..
-t
± ± ± ± ± ± ± ± ± ± ± ± ±
.1 .1 .7 .1 .7 .4 .7 .0 .1 .0 .0 .1 .0 ± .2
aValues are molesi100 mol ± SEM from four whole carcasses per sex each. bOnly major fatty acids are reported. The values for and C22.6 n.3 fatty acids were less than .1%. cPUFA = polyunsaturated fatty acids.
C20:5n.3. C22:5n.6.
(34.4 mol/100 mol), C 18l n.9 (26.4 mol/100 mol), and C 16:0 (24.1 mol/100 mol). The level of C 180 (6.8 mol/ 100 mol) and C20:4 (1.4 mol/100 mol) were low. Monoenes and PUFA together contributed 66.4 mol/100 mol of the total fatty acids. This result differs significantly from the values reported by Cambero et al. (3), in which C 16:0 contributed 40% of the total lipids and C 18:2n.6' 11 %. According to CamResults and Discussion bero et al. (3), monoenes and PUFA together Table 1 shows the lipid composition of the rabbit represented 39.1% of the total fatty acids. Cambero - -lea-r+-mbisGI s-tissbls-.----T--Rs-t-GtaWif)iG-GGRt€ Rt-Gf- r-at>bit-et--al.-(dj--a_oo-~a_G-e-t-aI-.----(-lJ-)-Gl-iQ-RGt-~sf>Gft-aR-Y-meat was 2.S and 3.0 g/100 g for male and females, values for C20:2, C20:3, and C204 in the total lipids for respectively. The overall mean total lipid was 2.7 ± rabbits of similar age, apparently because they .7 g/100 g. Cambero et al. (3), Lukefahr et al. (12), used packed column GC in their analysis. and Rao et al. (13) have reported Slightly higher Our results were comparable to those reported values (4.9 to 7% total fat). Our results were most by Lee and Ahn (11) except that we detected more comparable to the 2.0 to 3.9% fat reported by Holfatty acids such as C 15:0, C 16:l n.7, C 18:3n.6, C20:l n.g, C20:2 mes et al. (9) and Zegarska et al. (16). The proporn.6, and C20:3n .6 with a capillary column. The commertion of neutral lipids to glycerophospholipids were cial diet consumed by the rabbits contained S.S% 63 and 29% and 65 and 26% for male and female fat (data not shown), whereas Cambero et al. (3) rabbits, respectively. fed 2.S% fat. Rabbit meat had a lower content of Table 2 shows the fatty acid composition of the total saturated fatty acids, 33.7 mol/100 mol, when rabbit lean muscle total lipids. There were no sigcompared with beef (S1.S mol/100 mol), pork (42.9 nificant differences in fatty acid composition bemol/100 mol), and chicken (36.S mol/100 mol) muscle tissue according to Lee and Ahn (11). Surpristween the maie - and female rabbits. The overaii mean values indicated that lean muscle tissue of ingly, we did not observe the presence of signifithe total carcass contained predominantly C 18:2 n.6 cant levels of C20:5 n.3, C22:5 n.6, and C22 :6 n-3 in the total
LIPIDS OF RABBIT LEAN MUSCLE TISSUE lipids. Our result is consistent with those reported by Cambero et al. (3, 4), Lee and Ahn (11), and Rao et al. (13). It may well be that the levels of these PUFA were too low to be detected under the conditions of our analysis. Table 3 shows the overall fatty acid composition of the glycerophospholipids (GPE, GPC, GPI, GLPC, and SPH). The predominant fatty acids in combined male and female rabbits GPE were C,s:, n-9 (26.8 mol/100 mol), C'S:2 n- S (24.2 moV100 mol), and C20:4 n-S (20.3 moV100 mOl). Notably, the level of PUFA was high and significant (P<.05) when the female (48.5 moV100 mol) was compared to male (42.9 moV100 mol) (data not shown). The amount of C'so and C'S:2 n-S was also significantly different between males and females. The females contained 30.1 moV100 mol C'S:2 n-S compared with 18.3 mol/100 mol in the males. Cambero et al. (4) also observed that females contain a higher concentration of C,s:2n-s than males. In general, the total saturated fatty acids were higher in male than in female rabbits. For GPC there were no significant differences between male and female rabbits in fatty acid composition. The overall composition indicated that the predominant fatty acids were C's:o (36.1 moV100 mol), C'S:2 n-S (30.2 moV100 mol), and C,s:, n-9 (18.7 moV100 mOl). The total saturated fatty acid content was higher than the monoenes and
43
PUFA. Cambero et al. (4) reported the fatty acid composition of GPE and GPC. They were unable to separate the other glycerophospholipid classes in the solvent system they used for TLC. No C20:4 noS' C20:5 n-3' and C22:S n-3 values were reported for GPC by Cambero et al. (4). Our results for GPC were in agreement with those reported by Cambero et al. (4) for C's:o and C,s:, n-9 but contrary to values reported for C's:o and C'S:2 noS' Cambero et al. (4) reported a value of 15 moV100 mol for C,s:2n-s, which represents half of the value reported here (30.2 moV100 mol). The values of C20:4 n-S (3.5 moll 100 mol) and C'S:3 n-3 (trace) that they reported for GPE were significantly lower than the values of 20.3 and 1.3 mo1/100 mol, respectively, reported here. In fact, Cambero et al. (4) reported that with the exception of very small amounts of C20:" which were always less than .7 mol/100 mol, they could not detect unsaturated fatty acids longer than C'B in glycerophospholipids. The relative proportions of individual glycerophospholipids classes as calculated after GLC in the presence of an internal standard were: GPC (55.4%), GPE (22.2%), GPI (11.0%), GPS (5.3%), SPH (4.4%), and GLPC (1.7%). Cambero et al. (4) and Kuksis (10) have reported similar values of 40 to 66% for GPC and 17 to 22% for GPE. The level of C'B:2 n-S in GPI was significantly different P<.05 in males (10.8 moV100
TABLE 3. Composition (molesl100 mol) of glycerophospholipids of rabbit lean muscle tissue ab . Fatty acid c GPS GPE GPC GPI 1.5 ± .1 1.1 ± .4 C'4:0 .6 ± .1 C'5:0 17.7 ± 1.3 12.3 ± 1.5 36.1 ± .8 13.2 ± 1.1 C,s:o .8 ± .1 C,s:, n.7 12.6 ± 1.8 7.7±1.1 37.1 ± 2.6 41.0 ± 3.6 C,s:o 23.7 ± 2.3 26.8 ± 1.6 18.7 ± .6 16.3 ± 1.6 C,s:, n.9 16.2 ± 1.5 24.2 ± 2.6 30.2 ± .8 8.3 ± .7 C'S:2 n·S 1.3 ± .3 C'S:3n.3 C20:0 20.3 ± 2.9 25.2 ± 2.0 Trd 7.2 ± .8 C20:4 n-S C22:0 C24:0 C24:' n-9 Saturated 26.6 ± 1.4 43.8 ± 1.8 50.2 ± 1.7 60.2 ± 2.2 23.7 ± 2.3 Monoenes 27.6 ± 1.6 18.7 ± .6 16.3 ± 1.6 PUFA 16.2 ± 1.5 45.7 ± 1.2 37.4 ± 1.4 33.6 ± 2.0
GLPC 5.6 ± .6
SPH 2.7 ±
39.1 ± 1.0
26.7 ± 1.3
15.4 ± 1.5 27.4 ± 1.7 12.5 ± 1.0
44.2 ± 1.2 9.3 ± .9 4.8 ± .5 4.9 ±
60.1 ± 2.2 27.4 ± 1.7 12.5 ± 1.0
.1
.5
2.4 ± .2 2.2 ± .2 2.6 ± .3 83.3 ± 1.3 11.9 ± 1.1 4.8 ± .4
aValues are molesl100 mol ±SEM from four male and four female carcasses. bGPE =glycerophosphoethanolamine; GPC =glycerophosphocholine; GPI =glycerophosphoinositol; GPLC =glycerolysophosphocholine; SPH = sphingomyelin; and PUFA =polyunsaturated fatty acid. COnly major fatty acids are reported. drr = trace amounts, less than .1%. Values for C20:5n-3, C22:5n-s, and C22:Sn-3were also less than .1%.
AKOH ET AL.
44
TABLE 4. Variancecomponent analysis of rabbit lean muscle tissue fatty mol) vs females (5.8 mo1/100 mol). The predomiacid for among-litter and within-litter (residual) sourcesab . nant fatty acids in GPI were, as expected, C 180 (37.1 Variance component (%) moV100 mol), C20: 4 n. 6 (25.2 moV100 mol), C18:1 n.9 ':I ..... ,,1/100 ..... ,,1\ Clnrl r 11':1" "'1"'11/1 ()() Fatty acids Residual Litters \'1 c:: v.v v v16:0 \ vv ..,,,,1\ Total lipid Cambero et al. (4) were unable to detect GPI in (92.5) (7.5) 2.171 .177 C16:0 their preparations. (85.4) 1.581 .271 (14.6) C18:0 Except for C14:0, there were no significant differ(94.1) (5.9) .080 .005 C18:3n.3 (14.9) PUFA .640 (85.1 ) .112 ences in the fatty acid composition in male and GPE female rabbit GPS (Table 3). Stearic acid C 18:0 (41.0 (86.6) .098 (13.4) .638 C14:0 9;063 (89.9) 1.021 (11.1 ) C16:0 mol/1OO molT was the preaominant fatty acid. Sur(91.3) (8.7) .035 .003 C16:1 n -7 prisingly, only traces of C20:4 n.6 were observed in (83.2) (16.8) .579 .117 C18:3n-3 GPS. No significant differences were observed in aOnlyfatty acids significantly influenced (P<.05) by the litter source of fatty acid composition bett/Jsen male and female variation are pi6santad in the table. bGPE = glycerophosphoethanolamine; PUFA =polyunsaturated fatty rabbit GLPC. The major fatty acids were C 16:0 (39.1 acid. moV100 mol) and C18:1n.9 (27.4 mol/100 mol). Notably, there was no C20: 4 n.5 in the GLPC, indicating that the GLPC was derived from possible hydrolysis by phospholipase A2 at the Sn-2 position. Totion for total lipids percentage has been previously gether, C16:0 and C18:0 contributed approximately 71.0 reported by Lukefahr et al. (12). Further research moV100 mol of the total fatty acids in the rabbit involving larger samples of rabbits would be useful SPH. Saturated fatty acids represented 83.3 moll to confirm present experimental results. This report 100 mol of the total fatty acids. As with other animal represents the first detailed analyses of the inSPH, the characteristic C20:0' C22:0, C24:0, and C24:1 n.9 dividual phospholipid class fatty acid composition of fatty acids were observed in rabbit meat and comrabbit lean muscle tissue. Domestic rabbit meat pare well to those reported by Kuksis (10). There consumption has not increased in recent years. Increasing of C18:1n.9 content of rabbit lean muscle were significant differences between male and female rabbits in content of C14:0, C 18:0, and total tissue may have potential health benefit in view of monoenes (data not shown). Of all the the fact that diets high in monoenes have been glycerophospholipids, SPH had the lowest amount suggested to have cholesterol-lowering effect and of PUFA and the highest amount of saturated fatty needs to be explored through breeding programs. Of particular concern is the fact that little or no long acids. There were some dimethylacetals formed during FAME preparation. However, their values chain n-3 PUFA were detected in both the total _are oat rep.o.ded io this....p.aFP=eL.cr_ _ _ _ _ _ _ _...;I~ipids and glycerophospholipid classes of this rabbit Overall, there was marked heterogeneity in the bree-d-:- -- ---- - fatty acid composition of the glycerophospholipid classes. Glycerophosphocholine and GPE were the Literature Cited major glycerophospholipid classes in rabbit lean 1. Akoh, C. C., and R. S. Chapkin. 1990. Composition of mouse muscle tissue, representing 55.4 and 22.2%, peritoneal macrophage phospholipid molecular species. Lipids 25: 613. respectively. It should be noted that (Table 4) there 2. Akoh, C. C., and J . O. Hearnsberger. 1991. Effect of catfish and was significant among-litter variation in some fatty salmon diet on platelet phospholipid and blood clotting in healthy men. J . Nutr. Biochem. 2:329. acids, inferring genetic and(or) common environ3. Cambero, M. I., L. de La Hoz, B. Sanz, and J. A. Ordonez. 1991. Lipid mental causes. The sex of the rabbits also conand fatty acid composition of rabbit meat: Part 1. - Apolar fraction. Meat Sci. 29:153. tributed in part to the total variance. Specifically, in 4. Cambero, M. I., L. de La Hoz, B. Sanz, and J. A. Ordonez. 1991. Lipid total lipids, only C16:0, C18:0, C 18:3n.3, and total PUFA and fatty acid composition of rabbit meat: Part 2. - Phospholipids. Meat Sci. 29:167. were significantly influenced by the litter source, 5. Chapkin, R. S., S. D. Somers, and K. L. Erickson. 1988. Dietary whereas in GPE, only C 14:0, C 16:0, and C 16:1n. 7 were manipulation of macrophage phospholipid classes: Selective increase in dihomogammalinolenic acid. Lipids 23:766. affected. The exact cause(s) explaining this source 6. Dyerberg, J ., H. O. Bang, E. Stofferson, S. Moncade, and J. R.Vane. of variation cannot be elucidated and thus warrant 1978. Eicosapentaenoic acid and prevention of thrombosis and further investigation. Significant among-litter variaatherosclerosis. Lancet ii:117. IIIVII
I
IIIVI"
UIIU
n."".~
111'-'11
I
111'VI}o
LIPIDS OF RABBIT LEAN MUSCLE TISSUE 7. Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. BioI. Chem. 226:497. 8. Grundy, S. M. 1986. Comparison of monounsaturated fatty acids and carbohydrates for lowering plasma cholesterol. New Engl. J. Med. 314:745. 9. Holmes, Z. A., S. F. Wei, D. J. Harris, P.R. Cheeke, and N. M. Patton. 1984. Proximate composition and sensory characteristics of meat from rabbits fed three levels of alfalfa meal. J. Anim. Sci. 58:62. 10. Kuksis, A. 1985. Animal lecithins. In: B. F. Szuhaj and G. R. List (Ed). Lecithins. p 105. American Oil Chemists Society, Champaign, IL. 11. Lee, Y. C., and H. S. Ahn. 19n. Studies on lipids and proteins of rabbit meat. 1. Emphasis on lipid components of rabbit meat. Korean J. Nutr. 10:78.
45
12. Lukefahr, S. D., C. V. Nwosu, and D. R. Rao. 1989. Cholesterol level of rabbit meat and trait relationships among growth, carcass and lean yield performances. J. Anim. Sci. 67:2009. 13. Rao, D. R., C. B. Chawan, C. P. Chen, and G. R. Sunki. 1979. Nutritive value of rabbit meat. In: The Domestic Rabbit: Potentials, Problems and Current Research. p 53. Oregon State University Press, Corvallis, OR. 14. SAS. 1985. SAS User's Guide: Statistics (Version 5 Ed.). SAS Institute Inc., Cary, NC. 15. USDA. 1989. Composition of Foods, Raw, Processed, Prepared. Agriculture Handbook 8. ARS, USDA, Washington, DC. 16. Zegarska, Z., K. Markiewicz, and S. Smoczynski. 1979. Composition of fatty acids in muscle fat and depot fat of rabbits. Zesz. Nauk. Art. Olsz. Technol. Zywnosci. 15:167.