Hepatic Fatty Acid Profiles in Aflatoxin-Exposed Broiler Chickens'

Transcription

1 Hepatic Fatty Acid Profiles in Aflatoxin-Exposed Broiler Chickens' J. W. MERKLEY US Department of Agriculture, Agricultural Research Service, Poultry Research Laboratory, Georgetown, Delaware R. J. MAXWELL and J. G. PHILLIPS US Deparment of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 6 East Mermaid Lane, Philadelphia, Pennsylvania W. E. HUFF US Department of Agriculture, Agricultural Research Service, Veterinary Toxicology Research Laboratory, P.O. Drawer GE, College Station, Texas (Received for publication May 5, 1986) ABSTRACT This study was initiated to determine whether a specific fatty acid marker from hepatic lipids could be identified to confirm aflatoxin exposure in chickens. Male broiler chicks (24) were reared to 21 days of age on diets containing,, 1.25, 5., and 1. ppm aflatoxin. Live weights and organ weights differed significantly among the dietary groups. Total liver lipids increased significantly (P<.1) with increasing aflatoxin levels. Separation of extracted lipids into neutral and polar fractions established that this increase in hepatic lipid was accounted for almost entirely by increases in neutral lipid fractions. A gain of 117% occurred in the weight of neutral lipids as aflatoxin increased from to 1 ppm. In both neutral and polar lipid fractions subsequent chromatographic analysis of fatty acid methyl esters (FAME) showed linear increases in the monoenoic fatty acids with increasing aflatoxin. No single FAME from either lipid fraction was suitable for confirming aflatoxin exposure. (Key words: aflatoxin, liver lipids, broilers, fatty acids) 1987 Poultry Science 66:59-67 INTRODUCTION latoxins present in feed at levels that do not Incidence of aflatoxicosis and its contribution increase mortality or depress growth rates can to economic losses experienced in commercial effect physiological changes that severly predispoultry operations are difficult to assess. Unless pose poultry to the complications of stress and the severity results in a high rate of mortality opportunistic disease. Tung et al. (1971) found and classical symptoms have not been altered that ppm aflatoxin in feed was insufficient by secondary disease agents, aflatoxicosis may to affect growth rates of commercial broilers, not be suspected and feed may not be analyzed but the energy required to produce a bruise was to determine the presence of aflatoxins. Often lowered by 18%. Capillary fragility increased when feed samples are collected for anlaysis, and lateral shear strength of breast muscle dethe contaminated feed may have been consumed creased at this same dose level. Hamilton and or be otherwise unavailable, and aflatoxins can Harris (1971) indicated that health problems asno longer be recovered to confirm the incidence, sociated with aflatoxins are not the result of true It is particularly disconcerting to realize that af- aflatoxins but are usually syndromes caused by interacting stresses. They found that aflatoxinexposed chickens were more susceptible to infection with Candida albicans, to stress from cold, and to 1 % NaCl in their drinking water. Chickens Mention of a trade name, proprietary product, or spe- with aflatoxicosis, however, were less SUSCepticific equipment does not constitute a guarantee or warranty,,.,..,.,.,.,..., r,, by theus Department of Agriculture and does not imply ble to heat stess whlch these authors > attributed its approval to the exclusion of other products that may be to a lack of body fat. Giambrone et al. (1985) suitable. demonstrated that chickens exposed to aflatoxin 59

2 6 MERKLEYETAL. levels that did not reduce growth were deficient in cell-mediated immunity, as measured by a delayed hypersensitivity skin test. Miller and Wyatt (1985) examined chlortetracycline concentrations in plasma of chickens treated with low levels of aflatoxin and reported that the lowered chlortetracycline level resulted from a more rapid elimination of this drug from plasma possibly via the kidney. Apparently, not only are aflatoxin exposed broilers more susceptible to environmental stresses and other disease agents but also the usual means for treatment of these conditions may be impaired. Consequently, when aflatoxicosis is suspected, birds should be examined for presence of aflatoxicosis and its interaction with other factors. Aflatoxin is primarily a hepatotoxin, and aflatoxicosis results in enlarged livers with increased lipid content. Such observations have been reported by Hamilton and Garlich (1971), Doerr et al. (1983), and Miller and Wyatt (1985). Symptoms of aflatoxicosis can be altered by the presence of other agents, including other mycotoxins (Huff and Doerr, 1981), such that a diagnosis based upon gross observations of hepatic changes may be inaccurate. A method for identifying the presence of aflatoxicosis that is applicable in the presence of complicating factors is needed for accurate diagnosis. This study, therefore, was initiated to determine what levels of aflatoxin would result in any significant changes in hepatic fatty acids that conceivably could be used as a confirming diagnostic indicator. MATERIALS AND METHODS Male broilers (Ross x Arbor Acre) were obtained from a local hatchery. After selection for lack of obvious defects, the 24 day-old unvaccinated and undebeaked chicks were wingbanded, weighed, and randomly distributed to 24 sections of a Petersime battery. The wire floors of the electrically heated battery were covered with brown paper for the first 7 days to facilitate greater mobility of the chicks. Feed and water were provided ad libitum under continuous lighting, and water troughs were cleaned every 2 to 3 days. The basal diet was composed of a commercial corn-soybean meal starter mash free of antibiotics and coccidiostats. Graded levels of aflatoxin were added to obtain six experimental diets, a or control, and, 1.25, 2.5, 5., and 1 ppm aflatoxin. Each diet was randomly assigned to four battery sections, each containing 1 chicks. Aflatoxin was produced by a controlled fermentation of rice by Aspergillus parasiticus NRRL 2999 and prepared for addition to the basal diet as previously described by Huff and Doerr (1981). Individual birds were weighed, and feed consumption for each of the four sections of each of the six treatment groups was measured at 7-day intervals. All live weights were obtained on full-fed broiler chicks. The experiment was terminated at 21 days when the broiler chicks were killed by cervical dislocation. The spleen, left kidney, liver, and abdominal fat pad were removed from each chick and weighed. Liver was individually bagged, rapidly frozen, and stored at -35 C until analyzed. The dry column quantitative extraction method of Maimer and Maxwell (1981) for simultaneous separation of neutral and polar fractions of extracted lipids was modified for use with liver. Lipid fractions were extracted from randomly selected livers of the,, 1.25, 5., and 1 ppm treatment groups. A maximum of 2 g of minced liver tissue was ground in a mortar with 25 g granular anhydrous sodium sulfate after adding 1 ml antioxidant solution [ mg Tenox 5, Eastman Kodak Corp., Rochester, NY, in methylene chloride (DCM)] and then reground with addition of 25 g celite. This mixture was added to a glass chromatographic column containing a plug of glass wool and 1 g of calcium phosphate:celite (1:19). The dry column was wetted with DCM and the neutral lipid fraction eluted with 1 ml DCM. Subsequently, the column was charged with 15 ml of DCM:methanol (9:1) to recover polar lipids as a separate fraction. Each lipid fraction was condensed in a rotary evaporator with a 45 C water bath to remove solvents. After addition of 1 ml of antioxidant solution to the flask, the lipids were transferred to a 1 ml volumetric flask and brought to volume with hexane. A measured aliquot of these samples was removed for gravimetric determination of extracted lipid and the remaining solution stored in sealed vials with sodium sulfate under nitrogen in a freezer for subsequent analysis. Phospholipids in both the neutral and polar fractions were determined by the method of Vaskovsky et al. (1975). A minimum of 15 randomly chosen samples of both lipid fractions from the,, 1.25, and 5. ppm treatment groups were derivatized to their fatty acid methyl esters (FAME) following the methods outlined by Maxwell and Maimer (1983).

3 HEPATIC FATTY ACIDS WITH AFLATOXICOSIS 61 A Hewlett-Packard 588A, level 4, gas-liquid chromatograph equipped with a model 7672A automatic sampler system, flame ionization detector, and magnetic tape storage capability was used for FAME analysis. The procedure followed was an adaptation of the method of Maxwell and Marnier (1983). Methyl esters were separated on a fused silica capillary column (5 m X.25 mm i.d.) with a bonded stationary phase of SP-234 (Quadrex, New Haven, CT). Helium was the carrier gas at a flow of 1 ml/min, and ntirogen was the makeup gas at a flow of 3 ml/min. The temperature program employed was 15 C to 16 C at.5 /min, 16 C to 17 C at 1. /min, 17 C to 19 C at 1.5 /min,and the final temperature held for 25 min. Tenox 5, used to prevent oxidative changes and maintain integrity of the fatty acids, is a mixture of equal quantities of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Upon examination of the chromatographs, both BHA and BHT peaks could be mistaken for fatty acid methyl esters (FAME). The BHT eluted just before myristic acid and was well separated from any adjacent peaks. However, BHA eluted much later, after henicosanoic acid (21:), and unless careful attention is given to this peak during development of the temperature program, BHA may elute and be integrated with a FAME. It is recommended that only BHT be used as the antioxidant. The experimental data were analyzed by the general linear models procedure of the Statisitical Analysis System (SAS, 1982). Analysis of data for live weights, feed conversions, and organ weights was based on pen averages, whereas analyses of all lipid data were based on individual observations. Where treatment effects were significant, appropriate coefficients for unequally spaced aflatoxin concentration were generated using the Proc Matrix program (SAS, 1982), and data for each variable were tested using orthogonal contrasts for linearity and departure from linearity (quadratic and cubic effects combined). Higher orders of contrast were not of interest, and remaining degrees of freedom were not used to test the presence of other trends. Although both linear and nonlinear trends may have been significant, only the significant contrast of highest order irrespective of the level of significance is indicated in the tables. Duncan's multiple range test was used to locate differences among treatment means for discussion purposes and these results are alluded to in the results and discussion sections. Standard errors of the means are listed and can be used to determine where significant differences might exist. Statements of statistical significance were based on 5% probability. RESULTS Live weights and feed conversions for the six treatment groups are presented in Table 1. Weight was progressively depressed by increased aflatoxin concentrations, as compared with the controls, over the 21-day experimental period. At 7 days the 1 ppm group was significantly different, at 14 days groups above 1.25 ppm were significantly different, and at 21 days groups above ppm of aflatoxin were significantly different. The pattern was similar for feed conversions where feed conversion progressively increased over that of controls during the experimental period. At 14 days of age, groups receiving above 2.5 ppm were significantly different: at 21 days, groups above ppm were significantly different. For both live weights and feed conversions, where treatment effects were significant, there was a significant departure from linearity with increases in aflatoxin. Relative organ weights are presented in Table 2. Differences were significant (P<.1) among the treatment groups for all organs. The relative weights of the spleen, left kidney, and liver increased with increasing concentration of aflatoxin. The abdominal fat pad decreased with increasing concentrations of aflatoxin after reaching a maximum of 1.9% in the 1.25 ppm group, which was significantly (<.1) different from all other groups. Departures from linearity with increasing concentrations of aflatoxin were significant for all organs. Total liver lipids increased significantly with increasing aflatoxin concentrations (Table 3). Livers from the 1 ppm group contained 67% more lipid than livers from the control group. There was no difference between the total lipid content of livers from the 5 and 1 ppm groups. Data in Table 3 show that most of the increase in liver lipid with increasing aflatoxin concentrations resulted from increases of the neutral lipids rather than polar lipids. The neutral lipid fraction increased 117% from the control to the 1 ppm group. As for total lipids, there was no difference in the amount of neutral lipids between livers from the 5 and 1 ppm groups. There was essentially no increase in polar lipid with increasing aflatoxin concentration in the feed. The ppm aflatoxin treatment resulted in the greatest

4 62 MERKLEYETAL. TABLE 1. Effect of aflatoxin on the performance of male broilers from to 21 days of age Age Aflatoxin concentration in feed, ppm Contrast 2 (d) Values are means SEM Live weij ght (g) inversion (g feed/ ggain) * ** 2 Orthogonal contrasts were tested where treatment effects were significant; L = linear, DL = departure from linearity, quadratic, and cubic contrasts were combined. = eatment effects were not significant. *P<.5. **P<. ***P<.1. polar lipid concentration-the only significant effect in all treatments. The treatment effect for the phospholipid content of livers was not significant and averaged 25 mg/g over all treatment groups examined. Total lipids and the neutral Organ TABLE 2. Effect of aflatoxin on relative organ weight of male broilers at 21 days} * * * and polar lipid fractions all departed significantly from linearity with increasing concentrations of aflatoxin. The fatty acid profiles of the neutral and polar liver lipid fractions are presented in Tables 4 and Aflatoxin concentration in feed, ppm Contrasts 2 Spleen, mg/1 g Left kidney, mg/1 g Liver, g/1 g Abdominal fat, g/1 g ** ** ** 1 Values are means SEM. 'Orthogonal contrasts were tested where treatment effects were significant; L = linear, DL = departure from linearity, quadratic, and cubic contrasts were combined. = eatment effects were not significant. *P<.5. **P<.

5 HEPATIC FATTY ACIDS WITH AFLATOXICOSIS 63 TABLE 3. Effect of aflatoxin on the liver lipids of male broilers at 21 days. 1 Aflatoxin concentration in feed, ppm Contrasts 2 Total lipids wet tissue Neutral lipids wet tissue Polar lipids wet tissue Phospholipids, mg/g wet tissue Values are means SEM Orthogonal contrasts were tested where treatment effects were significant; L = linear, DL = departure from linearity, quadratic, and cubic contrasts were combined. = eatment effects were not significant. *P<.5. **P<. ***P<.1. 5, respectively. The identity of over 92% of the total fatty acids present in each fraction have been established. In both the neutral and polar lipid fractions, over 85% of their total fatty acid content are accounted for by five fatty acids, although only four of the principal fatty acids are the same in both fractions. In the neutral fraction from livers of the control group the principal fatty acids were palmitic (16:, 28.46%), palmitoleic (16:1, 5.18%), stearic (18:, 11.1%), oleic (18:lco9c, 32.22%), and linoleic (18:2«6, 9%). The polar fraction from the control livers contained the following principal fatty acids: palmitic (2.%), stearic (24.31%), oleic (14.14%), linoleic (18.54%), and arachidonic (2:4o>6, 9.6%). Of the principal fatty acids, only palmitoleic and linoleic in the neutral fraction and arachadonic in the polar fraction were unaffected by aflatoxin levels in the feed. In the neutral fraction stearic and arachidic (2:) acids increased linearly and significantly with increasing levels of aflatoxin. Stearic acid increased 22% from the control to the 5 ppm group. Arachidic acid increased 55% from the control to the 5 ppm group but was present in only minor amounts (<1%) in all treatment groups. Palmitic acid and several minor fatty acids (Peaks 4, 29, and 3) had significant linear decreases with increasing aflatoxin levels. Palmitic acid decreased 21% from the control to the 5 ppm group. The ratio of palmitic to stearic (16:/18:), two fatty acids with significant linearity, was not indicative of aflatoxin levels, and there were no differences among the treatment groups for this ratio. The ratio of the sum of the percentages of palmitoleic, stearic, and oleic to the percentage of palmitic methyl esters (lipo:mito) differed significantly (P<.1) and increased linearly with increasing concentrations of aflatoxin. The total identified saturates and co6 polyenes were not affected by the experimental treatments (Table 4). The significant (P<) linear increase in the total monoenes with increasing aflatoxin was not large and the control and 5 ppm group differed by only 1%. In the polar fraction, there was a significant linear increase in oleic, myristic (14:), and vaccenic (18:lco7c) acids with increasing aflatoxin levels. Unidentified Peaks 7, 45, and 51 also had significant linear increases with increasing treatment levels of aflatoxin. The percentages of linoleic and acids with peak numbers 3, 32, 49, and 54 had significant linear decreases with the increase in treatment levels of aflatoxin. Other individual fatty acids were either not affected by the dietary treatments or the changes significantly departed from linearity. As in the neutral fraction, total monenes in the polar fraction also increased significantly in a linear manner with aflatoxin levels. The increase from the control to the 5 ppm livers was 26%. Total saturates and co3 polyenes were not affected by the aflatoxin treatments and the ratio of palmitic to stearic (16:/18:) significantly departed from linearity. No gas chromatographic peak was de-

6 64 MERKLEY ET AL. tected in either fraction that appeared or disappeared with the presence of aflatoxin in the feed at any treatment level. It was concluded that the chromatographic analysis of samples from the 2.5 and 1 ppm treatment groups was not necessary for interpretation of the data and was therefore not conducted. DISCUSSION Results (Table 1) indicate that between 1.25 and 2.5 ppm aflatoxin was required before the weight of broiler chickens at 21 days of age was significantly depressed. The lack of linearity observed in the significant response of live weight and feed conversion with increasing concentra- Peak no : 2 14: : : : 15 18:lco9c 17 18:lu;7c :2CJ6 21 2: 22 18:3CJ : :3u>6 37 2:4o> :4co :6CJ3 55 Saturates Monoenes 16:/18: Lipo:Mito 5 u>6 polyenes TABLE 4. Hepatic lipid fatty acid methyl esters from aflatoxin exposed broilers at 21 days (Neutral fraction)* Aflatoxin concentration in feed, ppm Contrasts * * * L* j *** * f * * # 1 Values, expressed as percentages of the total fatty acids, are means SEM. 2 Peak numbers are assigned in order of retention times. If peak has been identified the carbon chain length, number and position of double bonds are given. 3 ace, amount detected <.1% in average of duplicates. "Orthogonal contrasts were tested where treatment effects were significant; L = linear, DL = departure from linearity, quadratic, and cubic contrasts combined. = eatment effects were not significant. s Sum of the percentages for 16:1, 18:, and 18:1 divided by the percentage of 16:. *P<.5. **P<. ***P<.1.

7 HEPATIC FATTY ACIDS WITH AFLATOXICOSIS 65 TABLE 5. Hepatic lipid fatty acid methyl esters from aflatoxin exposed broilers at 21 days (Polar fraction) 1 Aflatoxin concentration in feed, ppm Peak no Contrasts : 2 14: 6 16: CJ9C lu>7c u> : 22 18:3u) : :3w6 37 2:4w :4w :6w3 Saturates Monenes 16:/18: Lipo:Mito 5 UJ6 polyenes t * t * Values, expressed as percentages of the total fatty acids, are means SEM * * DL'** * ' * ' *» * * 2 Peak numbers are assigned in order of retention times. If peak has been identified the carbon chain length, number and position of double bonds are given. 'ace, amount detected <.1% in average of duplicates. 4 Orthogonal contrasts were tested where treatment effects were significant; L = linear, DL = departure from linearity, quadratic, and cubic contrasts combined. = eatment effects were not significant. 5 Sum of the percentages for 16:1, 18:, and 18:1 divided by the percentage of 16:. *P<.5. **P<. ***P<.1. tions of aflatoxin is in agreement with results reported in the literature. In earlier aflatoxin studies, Smith and Hamilton (197) concluded that a threshold level was required before body and organ weights were significantly affected. Recently Doerr et al. (1983) discussed differences between the levels of aflatoxin necessary to affect the live weights of broilers in two trials of a study. Thresholds of less than 5 ppm in one trial and 2.7 ppm in another trial were reported for 7-week-old broilers. Although those authors suspected the influence of a secondary factor that contributed to an apparently lower threshold level in the first trial, none could be identified. Not only are aflatoxin-exposed broilers underweight but this reduced weight gain would also be significantly more costly under commercial conditions where less efficient feed utilization would further contribute to the economic loss. The increase of the spleen, left

8 66 MERKLEYETAL. kidney, and liver and the decrease of the abdominal fat pad relative to body size found in this study (Table 2) are typical of the symptoms usually associated with aflatoxicosis (Huff and Doerr, 1981). The abdominal fat pads from chickens in the 1 ppm group appeared to be composed mostly of connective tissue and possibly represent a minimum possible weight. Under the conditions of this study, these symptoms of aflatoxicosis would indicate that no major complicating factors were present, and valid conclusions could be drawn from further analysis. Aflatoxins are classified as hepatotoxins, and in poultry as in other animals, the liver is the most sensitive tissue to this toxin (Ciegler, 1975). In the healthy chicken, de novo fatty acid synthesis takes place primarily in the liver and the primary function of broiler adipose tissue appears to be lipid storage rather than lipid synthesis (Nir and Lin, 1982; O'Hea and Leveille, 1969). The accumulation of lipid material in the liver observed in this study was accounted for exclusively as neutral lipids that were probably in the form of triglycerides. The increase in weight of the neutral lipids observed in this study, although indicative of liver injury, is not a uniquely specific resonse to aflatoxins. The most common response of the liver to injury is an abnormal accumulation of fat, largely, if not exclusively triglyceride, in the parenchymal cells (Witting, 1973). It is well-established that dramatic changes in the activities of liver enzymes involved in carbohydrate, amino acid, and lipid metabolism take place during the first 3 to 4 weeks posthatch. In the chicken, lipogenesis is minimal at Day 1 and rapidly increases during the first week to a maximum level and then after 3 weeks begins a gradual decline until reaching maturity (Raheja et al., 1971). If exposure to aflatoxins had any direct effect upon the pattern of fatty acids present in hepatic lipid fractions, poultry should be most susceptible during the first 3 weeks posthatch and any changes should be readily detectable at 21 days. The fatty acid profiles (Tables 4 and 5) showed no unusually large magnitude of change for any fatty acid among the treatment groups for either the neutral or polar lipid fraction. Reasons for the statistically significant changes in the fatty acids, including the essential fatty acids, in relation to aflatoxin exposure cannot be explained at this time. In both lipid fractions, the total monoenes had a significant linear increase with increasing levels of aflatoxin. This information would be of little value in attempting to confirm aflatoxin exposure, because fatty acid patterns in poultry tissue are significantly affected by the composition of the diet (Marion and Woodroof, 1963). In the diagnostic laboratory, carcasses of control and exposed broilers would not be available for comparisons. A method using fatty acid patterns or a specific marker to confirm the exposure of broilers to aflatoxin would have to be independent of dietary considerations. Although we observed a significant increase in accumulated lipid material, particularly neutral lipids, in hepatic tissue during aflatoxicosis, only relatively small changes in the pattern of fatty acids were found. Some of these small changes observed in the fatty acid patterns among the treatment groups could possibly be accounted for by the effects of feedback mechanisms initiated by the abnormal increase in hepatic lipids. The total lipid analysis would appear to support the conclusions reached by Tung et al. (1972) that lipogenesis is not affected by aflatoxins while mobilization of lipids from the liver is blocked. The primary fatty acids produced by de novo lipogenesis are palmitic, palmitoleic, stearic, and oleic. Palmitic acid is a product of fatty acid synthetase activity and palmitoleic, stearic, and oleic acids result from mitochondrial and microsomal elongation and desaturation. The significant (P<.1) decrease in palmitic and the significant (P<.1) increase in the ratio of these fatty acids (lipo:mito) in the neutral fraction would indicate that lipogenesis is reduced with aflatoxin exposure. This later conclusion would be in agreement with that of Donaldson et al. (1972). The treatment of ppm aflatoxin does not conform to the general trend; remaining treatment levels tended to yield linear points. Whether this is an anomaly of this single study or a predictable result of aflatoxin exposure cannot be answered at this time. It is obvious that this subject warrants further investigation. ACKNOWLEDGMENTS The authors gratefully acknowledge the cooperation and technical assistance of J. C. Richardi and Peter Vail. REFERENCES Ciegler, A., Mycotoxins: occurrence, chemistry, biological activity. Lloydia 38: Doerr, J. A., W. E. Huff, C. J. Wabeck, G. W. Chaloupka,

9 HEPATIC FATTY ACIDS WITH AFLATOXICOSIS 67 J. D. May, and J. W. Merkley, Effects of low level aflatoxicosis in broiler chickens. Poultry Sci. 62: Donaldson, W. E., H. T. Tung, and P. B. Hamilton, Depression of fatty acid synthesis in chick liver (Gallus domesticus) by aflatoxin. Comp. Biochem. Physiol. 41B: Giambrone, J. J., U. L. Diener, N. D. Davis, V. S. Panangala, and F. J. Hoerr, Effects of aflatoxin on young turkeys and broiler chickens. Poultry Sci. 64: Hamilton, P. B., and J. D. Garlich, Aflatoxin as a possible cause of fatty liver syndrome in laying hens. Poultry Sci. 5:8-84. Hamilton, P. B., and J. R. Harris, Interaction of aflatoxicosis with Candida albicans infection and other stresses in chickens. Poultry Sci. 5: Huff, W. E., and J. A. Doerr, Synergism between aflatoxin and ochratoxin A in broiler chickens. Poultry Sci. 6: Marion, J. E., and J. G. Woodroof, The fatty acid composition of breast, thigh, and skin tissues of chicken broilers as influenced by dietary fats. Poultry Sci. 42: Manner, W. N., and R. J. Maxwell, Dry column method for the quantitative extraction of simultaneous class separation of lipids from muscle tissue. Lipids 16: Maxwell, R. J., andw. N. Manner, Systemicprotocol for the accumulation of fatty acid data from multiple tissue samples: tissue handling, lipid extraction and class separation, and capillary gas chromatograph analysis. Lipids 18:453^159. Miller, B. L., and R. D. Wyatt, Effect of dietary aflatoxin on the uptake and elimination of chlortetracycline in broiler chicks. Poultry Sci. 64: Nir, I., and H. Lin, The skeleton, an important site of lipogenesis in the chick. Anim. Nutr. Metab. 26:1-15. O'Hea, E. K., and G. A. Leveille, Lipid biosynthesis and transport in the domestic chick {Gallus domesticus). Comp. Biochem. Physiol. 3: Raheja, K. K., J. G. Snedecor, and R. A. Freedland, Activities of some enzymes involved in lipogenesis, gluconeogenesis, glycolysis and glycogen metabolism in chicks (Gallus domesticus) from day of hatch to adulthood. Comp. Biochem. Physiol. 39B: SAS User's Guide: Statistics, SAS Inst., Cary, NC. Smith, J. W., and P. B. Hamilton, 197. Aflatoxicosis in the broiler chicken. Poultry Sci. 49: Tung, H. T., W. E. Donaldson, and P. B. Hamilton, Altered lipid transport during aflatoxicosis. Toxicol. Appl. Pharmacol. 22: Tung, H. T, J. W. Smith, and P. B. Hamilton, Aflatoxicosis and bruising in the chicken. Poultry Sci. 5: Vaskovsky, V. E., E. Y. Kostetsky, and I. M. Vasendin, A universal reagent for phospholipid analyses. J. Chromatogr. 114: Witting, L. A., Fatty liver induction: Effect on ethionine on polyunsaturated fatty acid synthesis. Biochim. Biophys. Acta 296: