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Plasma Free Fatty Acid and Neutral Lipid Concentrations in Immature, Laying and Broody Turkey Hens
1154
R. A. GUILL AND K. W. WASHBURN
E. W. Glazener, 1958. The heritability of body weight, gain, feed consumption and feed conversion in broilers. Poultry Sci. 37: 862-869. Timon, V. M., and E. J. Eisen, 1970. Comparisons of ad libitumand restricted feeding of mice selected and unselected for post-weaning gain. I. Growth, feed consumption and feed efficiency. Genetics, 64: 41-57.
Warwick, E. J., 1958. Fifty years of progress in breeding beef cattle. J. Animal Sci. 17: 922-943. Wilson, S. P., 1969. Genetic aspects of feed efficiency in broilers. Poultry Sci. 48: 487-495. Winters, L. M., and J. McMahon, 1933. Efficiency variation in steers. Minnesota Agr. Exp. Sta. Tech. Bull. 94: 1-28.
W A Y N E L . BACON, MARGERY A . MUSSER AND KEITH I. BROWN
Department of Poultry Science, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691 (Received for publication September 24, 1973)
ABSTRACT Free fatty acid (FFA) concentrations in blood plasma from immature, laying and broody turkey hens were re-examined following removal of phospholipids. A mean value of .21 ± .02 (A. equiv./ml. plasma of FFA was found for immature hens and as they were stimulated into egg production, and no systematic increase was noted. Broody hens had the same concentration as laying and immature hens. Neutral lipid levels (mainly triglyceride) increased from about 3.5 mg./ml. plasma to about 18-25 mg./ml. plasma as the hens reached sexual maturity, then plateaued until a broody period began when they dropped to concentrations comparable to those in immature hens. No correlation was found between FFA and neutral lipid concentrations. Although FFA concentration is much lower than previously reported, 14% of the total daily amount cleared was calculated to be sufficient to account for egg yolk neutral lipid. The remaining 86% would be available for other metabolic functions. POULTRY SCIENCE 53: 1154-1160, 1974
INTRODUCTION
P
LASMA free fatty acid (FFA) levels in chickens have been reported to increase from about 0.3 JJL. equiv. per ml. in non-laying pullets to about 1.0 to 2.0 |JL. equiv. per ml. in laying hens (Heald and Badman, 1963). In this study, FFA levels were determined by the titrimetric method of Dole and Meinertz (1960). A similar study has recently appeared for turkeys (Bajpayee and Brown, 1972). Prior to exposing immature birds to stimulatory lighting, plasma FFA concentration was 1.14 ± .20 |JL. equiv. per ml. After 21 days of stimulatory lighting, FFA concentration had almost doubled to 2.15 ± .11 (ju equiv. per ml. These authors used the method of Kvam
et al. (1964) to determine plasma FFA. This method is based on forming the copper salts of fatty acids (FA) and then colorimetrically determining the amount of copper associated with the fatty acids after they are extracted. The effect of fasting and gonadotrophin injection on plasma FFA levels was studied by Heald and Rookledge (1964). They observed that fasting caused an increase in plasma FFA in normal cocks, but a decrease in plasma FFA in laying hens when initial FFA levels were high and an increase when initial levels were low. Their method of determining FFA was based on the titrimeteric method of Dole and Meinertz (1960). When pituitary powder was injected during four-day starvation of laying hens, plasma FFA levels were maintained, as were plasma total lipids.
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Plasma Free Fatty Acid and Neutral Lipid Concentrations in Immature, Laying and Broody Turkey Hens
PLASMA FATTY ACID AND LIPID
During the course of FFA metabolism studies in laying turkey hens in this laboratory, the concentration of plasma FFA was found to range between .06 to .20 |x. equiv. per ml. In these determinations, PL's were removed by chromatography on silica gel and the FFA's extracted by alkaline ethanol (Borgstrom, 1952) before being quantitated by the method of Novak (1965). This method of FFA quantitation is chemically similar to that of Kvam et al. (1964) except that the cobalt instead of copper salts of the FFA's are formed. Since these results indicated much lower levels of plasma FFA's in laying turkey hens than previously reported, it was decided to do a more complete study of plasma FFA concentration in non-laying and laying turkey hens. In addition, the amount of plasma neutral lipid was determined. MATERIALS AND METHODS Turkeys. Experiment 1. Blood was collected from several laying hens and pooled. Experiment 2. Six young female turkeys
were housed in a floor pen when 24 weeks old, on September 21. They were exposed to natural daylight until February 21, when they were given 14 hours of light per day at about 50 lux intensity. Blood samples were taken about 3 times per week, starting two days before giving stimulatory lighting. About two weeks after full production was attained in all birds, samples were taken at about weekly intervals (see Tables 2 and 4). The birds were moved from the floor pen to cages (48 x 66 x 55 cm.) on March 6, or 3 days before the first egg was laid. Blood Samples. An 8-9 ml. sample of blood was drawn from each bird on each sampling date. The samples were placed in centrifuge tubes containing sufficient heparin to inhibit clotting and the plasmas were collected after centrifugation. The plasmas were then frozen and stored at -20° until analyzed within 2 months. Analyses. Experiment 1. Plasma was pooled from several laying turkeys and 1.0 ml. extracted either with or without added palmitic acid (0.40 9. equiv.) by the method of Trout et al. (1960). Iso-octane was substituted for heptane (Eaton et al., 1969). After extraction, two aliquots with and two without added palmitic acid were set aside for FFA determination by the method of Novak (1965). PL's were removed from the remainder of the extracts by silica gel chromatography (Bio Sil HA, BioRad, Richmond, California) as follows. One and one tenth g. of silica gel were weighed and covered with hexane:diethyl ether (H:D), 1:1. Columns (top 1/2 of a 100 ml. volumetric pipet, i.d. 0.6 cm.) were plugged with glass wool, filled with the silica gel slurry and then allowed to drain dry. The plasma extracts were then added to the columns in 1.0 ml. H:D, 1:1. The sample containers were then washed 2x with additional 1.0ml. amounts of H:D, 1:1 and added to the columns when dry. After this, 7.0 ml.
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If pituitary powder was not injected, both plasma FFA and total lipids levels fell. Neither of the above methods for plasma FFA determination give absolute specificity for FFA concentration, as phospholipids (PL) may interfere if present in either large and/or varying amounts (Dole and Meinertz, 1960; Kvam et al., 1964). It is well known that when a hen enters egg production a lipophosphoprotein complex (LPP) of constant composition can be precipitated from the plasma. LPP is not detected in the plasma of non-laying hens or cocks (Mclndoe, 1959). Furthermore, LPP is composed of 80% lipid, of which about 25% is PL. This indicates large changes in the amounts of PL when non-laying and laying female or when fasting vs. full fed laying female plasmas are compared. If the PL's are not removed prior to FFA determination by the above methods, the levels of circulating FFA's may be overestimated.
1155
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W. L. BACON, M. A. MUSSER AND K. I. BROWN
of Novak (1965). Eighteen to 24 plasma samples were run in each analysis. The standard curve for the FFA's was determined for each analysis with 0, 0.02, 0.04 and 0.06 |x. equiv. of palmitate. To each standard, an aliquot from a blank column was added equivalent to the sample aliquot to compensate for the small amount of background color attributed to column washing by the eluting solvents. FFA concentrations were adjusted to 100 percent recovery. Actual radioactivity recovery was 95 ± 1% (s.e.). RESULTS Experiment 1. It is clearly seen in Table 1 that removal of PL's by the relatively simple Bio Sil HA chromatography step reduces detectable FFA levels by about 75%, while not reducing the recovery of added FFA. After isolation of the FFA's by alkaline ethanol partition, the FFA concentrations for samples with and without added FFA were nearly the same as these respective samples before solvent partition. These data indicate that the high FFA levels previously reported for both laying turkeys (Bajpayee and Brown, 1972) and chickens (Heald and Badman, 1963; Heald and Rookledge, 1964) were probably due to a non-specific overestimation caused by the presence of PL's in their samples. The relatively high concentrations of plasma FFA present in laying in comparison to nonlaying and fasted hens previously reported are probably due to increases in PL con-
TABLE 1.—Free fatty acid concentrations determined on Trout extract, extract after Bio Sil HA chromatography and extract after both chromatography and alkaline ethanol partition
With added FA' Without added FA Difference
'Added 0.40 (x. equiv. palmitic acid. equivalent/ml. plasma ± s.e. Only one sample determined.
2 \x. 3
Trout extract
After Bio Sil chromatography
After chromatography and solvent partition
1.23 ± .042 0.91 ± .04 .32
.68 ± .01 .263 .42
.66 ± .03 .22 ± .01 .44
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of H:D, 1:1 was added to each column and they were allowed to run dry. The tips of the columns were then washed with diethyl ether. After drying and resuspension, an aliquot of each extract was taken for FFA determination. FFA's were extracted from the above purified extracts by partitioning between iso-octane and alkaline ethanol by the method of Borgstrom (1952). An aliquot was then taken in duplicate for FFA determination. Experiment 2. The 1.0 ml. plasma samples were extracted by the method of Trout et al. (1960), using iso-octane instead of heptane and omitting the 0.05% aqueous H 2 S0 4 wash. Since the extracts were to be passed through columns of silica gel the upper layer was completely removed and the lower layer washed 2 x with additional iso-octane and the upper layers combined. These extracts were taken to dryness in a vacuum over at 60° and then chromatographed on Bio Sil HA as in Experiment 1. A tracer dose (6,000 to 7,000 C.P.M.) of 1-C14-Palmitic Acid was added to each sample prior to extraction. The column eluates were collected into tared scintillation vials, the eluting solvents evaporated under vacuum at 60° and the vials then reweighed to determine the amount of neutral lipid, mainly TG and cholesterol, extracted from each sample. After weighing, H:D, 1:1 was added and duplicate aliquots removed to determine both radioactivity recovery and FFA concentration by the method
PLASMA FATTY ACID AND LIPID
1157
TABLE 2.—Free fatty acids and neutral lipids for the various days post stimulatory lighting
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of egg production. The one high mean value at day 14 is probably associated with either the stress of moving the birds from a floor Days FFA ± s.e. Neutral lipids pen to cages or lowered feed consumption u.. equiv./ post 'og.o just prior to initiation of egg production, or lighting ml. plasma mg./ml. ± s.e. mg./m both. Unpublished results from this laborato3.8 .58 ± .06 -2 .15 ± .02 3.3 .52 ± .06 .22 ± .03 0 ry and the report of Meyer et al. (1970) show 4.8 .68 ± .07 .18 ± .02 3 decreased feed consumption at the time of 7.2 .86 ± .04 .19 ± .02 5 10.2 1.01 ± .06 .22 ± .02 7 first egg in turkeys and chickens, respec10.5 1.02 ± .07 .17 ± .01 9 tively. Feed restriction may cause increased 18.5 1.27 ± .04 .17 ± .02 12 14.1 1.15 ± .05 .36 ± .08 14' FFA levels in normal cocks (Heald and 12.2 1.09 ± .06 .29 ± .04 162 Rookledge, 1964) or male turkeys (Bacon and 16.9 1.23 ± .06 .33 ± .08 19 18.7 1.27 ± .07 .24 ± .05 21 Musser, unpublished data). 18.6 1.27 ± .04 .26 ± .07 24 Neutral lipids (mg./ml. plasma) were 17.6 1.24 ± .04 .23 ± .04 26 14.6 1.16 ± .04 .23 ± .05 30 transformed to their log I0 values for analysis 19.3 1.28 ± .05 .14 ± .02 35 of variance, to normalize the variance about 23.7 1.37 ± .04 .17 ± .02 42 26.6 1.42 ± .04 .18 ± .05 49 the means. Table 2 gives both the transformed LSD 3 values ± s.e. and the converted mean values. 26 .22 P < .05 .: ' Birds moved from floor pen to cages on day All comparisons were made using the trans132post lighting. formed values. It is readily apparent that the First eggs were laid from day 16 to 24 post amount of neutral lipids increases during the lighting. 'Method of Tukey (Steel and Torrie, 1960). initial phase after stimulatory lighting, until egg production begins, and then is maintained centration rather than to increases in FFA at this relatively high level as long as the individual hen remains in egg production. concentration. To determine whether the However, a considerable range of levels eiFFA concentration did increase, the second ther between laying hens on a given date experiment was conducted. or within individual laying hens on different dates was found (Table 3). The upper values Experiment 2. The data from 2 days prewere found to be about threefold higher than and the first 49 days post stimulatory lighting the lower values. These data agree with the are given in Table 2. Analyses of variance plasma total lipid data presented by Heald are presented in Table 3. The first eggs from and Badman (1963). all 6 birds were laid from days 16 to 24 (mean 19.0 ± 1.2 days), which was after the birds were moved from the floor pen to cages on TABLE 3.—Analyses of variance of FFA and log, day 13. neutral lipids The FFA level on day 14, or the first day Neutral after moving, was greater (P < .05) than on lipids days - 2 (before lighting) and 35, when all FFA (logio) 1 ms ms Source df of the birds were in full production (Table 2). No significant increase up to time of first Days post .023544* .069786** lighting 16 egg and during initial egg production was .454407** Birds .034460* 5 Residual .014434 80 .010890 noted. This is in contrast to the findings of 1 The mean squares associated with post lighting Heald and Badman (1963) and Bajpayee and and birds were both tested for significance (* = Brown (1972) both of whom reported in- P < .05; ** = P < .01) using the residual mean creases in FFA concentration during initiation squares.
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W. L. BACON, M. A. MUSSER AND K. I. BROWN
TABLE 4.—Free fatty acid and neutral lipids in laying and broody turkey hens Neutral lipids Days post lighting
n
FFA ± s.e. JJL. equiv./ ml. plasma
4 5 4 3 2 3 3
58 61 65 69 75 82 89
2 1 2 3 4 3 3
Broody .41 ± .29 .18 ± .28 ± .18+ .21 ± .16 ±
An overall correlation coefficient with treatment effects absorbed was calculated between FFA and neutral lipid levels. The value of this correlation was -.011 (d.f. = 100) and was not significant (P > .05). This indicates no relationship between plasma FFA and neutral lipid levels as the hens came into and during the first four weeks of production. This is in contrast to the findings of Heald and Badman (1963) and Heald and Rookledge (1964). After 49 days post lighting, broodiness became evident in various hens on various dates. Broodiness is defined as a period in which no eggs are laid for five or more days. Table 4 compares FFA and neutral lipid levels from laying vs. broody hens. It is apparent from this data that there is no sustained difference in FFA level between the laying and broody hens, while neutral lipid levels decrease to levels at or below those present before or on the day of exposing the same birds to stimulatory lighting. The mean value of 0.41 ± .17 p.. equiv./ml. on day 58 in the broody hens was the highest mean observed during the experiment. However, only two hens were broody on this date, one of which assayed at 0.54 \x. equiv./ml. plasma. Individual values up to 0.76 JJU equiv./ml.
hens .01 .02 .06 .01 .01 .04 .02 hens .17 .01 .10 .01 .01 .03
mg./ml.
.02 .02 .05 .10 .02 .03 .09
26.3 21.8 19.8 25.6 30.2 31.4 27.1
.64 ± .01 .45 .58 ± .24 .49 ± .10 .44 ± .06 .37 ± .02 .36 ± .03
4.4 2.8 4.4 3.3 2.8 2.3 2.3
1.42 1.34 1.29 1.38 1.48 1.49 1.41
± ± ± ± ± ± ±
were occasionally noted throughout the experiment. The cause of these high values is unknown and the occurrence was random. DISCUSSION The major finding of the present work is that there is no difference in FFA concentration among immature, laying and broody turkey hens. This is in contrast to the findings of Heald and Badman (1963) and Bajpayee and Brown (1972) for chickens and turkeys, respectively. The increases reported by the above workers can be attributed to the interference of PL in the methods used to determine FFA concentration. In addition, no correlation was found between levels of FFA and neutral lipids in plasma, which is in contrast to the results of Heald and Badman (1963) and Heald and Rookledge (1964) who demonstrated increased levels of FFA coinciding with increased levels of total plasma lipids (neutral lipids + PL's). This correlation can also be ascribed to the lack of specificity in the methods used by the above authors to determine plasma FFA levels. The mean plasma levels of FFA and neutral lipids among the hens while they were laying was .21 ± .02 p.. equiv./ml. and 21.8 ± 1.3 mg./ml., respectively. Plasma neutral lipids
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58 61 65 69 75 82 89
Laying .21 ± .20 ± .26 ± .14 ± .10 ± .16 ± .15 ±
log10 mg./ml. ± s.e.
1159
PLASMA FATTY A C I D AND LIPID
laboratory (Bacon and Musser) indicate similar turnover times for plasma FFA in laying turkey hens. This value (.14) would indicate the ratio of plasma FFA directly entering plasma LPP neutral lipid TG-FA if LPP neutral lipid TG-FA came directly from plasma FFA and only 5.45 g./day LPP TG-FA is synthesized. Both of these assumptions are highly unlikely. It has been reported recently by Bacon (1973) that plasma neutral lipid TG-FA in laying turkey hens has a turnover time of .43 days instead of 1.1 days. If this value is used to estimate the total amount of plasma neutral lipid removed per day, about 6.4 g. -5- .43 day or 14.8 g./day would be removed. This value is 8.4 g. greater than that calculated from the estimate of 1.1 day for turnover time. This 8.4 g./day is equal to 5.0 mg./min. of LPP TG-FA being removed from the plasma in addition to what is removed by the ovary. This LPP TG-FA could either be removed to the adipose tissue and stored as tissue lipid or re-enter the plasma as FFA. The removal rate of plasma FFA as stearate is 20.4 mg. H- .83 min. or 24.7 mg./min. If we assume that the above 5.0 mg./min. of LPP TG-FA enters the plasma as FFA, then 20% of the plasma FFA could come directly from LPP TG-FA. Calculations such as those above depend upon accurately measuring the concentrations of various plasma constituents and their rates of loss from the plasma and any possible recycling. In the present experiments, we feel that the estimate for plasma FFA has been more accurately made than in previous reports. We are currently studying the kinetics of FFA and LPP TG-FA and hope to report the results of these experiments in the near future. REFERENCES Bacon, W. L., 1973. Turnover time and metabolic clearance rates of very-low-density lipoproteins in
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decreased to 3.3 ± .4 mg./ml. in the broody hens. The difference in the concentration of neutral lipids in the hens when they were laying vs. when they were broody is 18.5 mg./ml. and is probably associated with the LPP complex of Mclndoe (1959). If the plasma volume is assumed to be 4.6% of body weight (McCartney, 1952), which is about 7.5 kg. for this strain, the plasma volume is 345 ml. Plasma content of neutral lipids and FFA is then 6.4 g./hen and 72 JJL. equiv./hen, respectively. If FFA content is multiplied by the molecular weight of stearate (284), an estimate of the plasma FFA content as stearate is obtained. With the present data, this estimate is 20.4 mg./hen. If a hen is laying eggs with 30 g. yolks and each yolk is 33% lipid (Gilbert, 1971), she is producing 10 g. lipid per day as egg yolk. Of this, about 17% is PL, so she is producing about 8.3 g. of neutral lipids per day if she lays at an intensity of 100%. In the present experiment, the six hens laid at an intensity of 70 ± 2%, so their rate of production of neutral lipids would be about 5.8 g./hen/day. When 6.4 g. neutral lipid/hen in the plasma is divided by 5.8 g. neutral lipid/hen/day lost as egg yolk, the value 1.1 days indicates the turnover time in days for the plasma neutral lipids if they are removed only by follicles in rapid development, and exit the bird as egg yolk. If total plasma content of neutral lipids is assumed to be 95% triglycerides (TG) and 90% of the TG is FA, the weight of plasma TG-FA associated with LPP would be 5.45 g. If it is assumed that all of the neutral lipid TG-FA is derived from plasma FFA, and that all plasma FFA enters TG, the plasma FFA would turn over at a rate 5.45 g./l.l day/.0204 g. or 243 times per day, or once each 5.9 minutes. The turnover time of plasma FFA calculated from the data of Heald and Badman (1963) in laying chicken hens is .83 minutes, which is .14 times as large as that calculated above. Unpublished data from this
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W. L. BACON, M. A. MUSSER AND K. I. BROWN
gonadal hormones, gonadotrophins, and thyroxine on plasma free fatty acids in the domestic fowl. J. Endocrinol. 30: 115-130. Kvam, D. C , J. G. Schmidt, D. A. Riggilo and D. G. Gallo, 1964. Colorimeteric microdetermination of plasma free fatty acids. J. Pharmaceutical Sci. 53: 988-989. McCartney, M. G., 1952. Total blood and corpuscular volume in turkey hens. Poultry Sci. 31: 184-185. Mclndoe, W. M., 1959. A lipophosphoprotein complex in hen plasma associated with yolk production. Biochem. J. 72: 153-159. Meyer, G. B., S. W. Babcock and M. L. Sunde, 1970. Decreased feed consumption and increased calcium intake associated with pullet's first egg. Poultry Sci. 49: 1164-1171. Novak, M., 1965. Colorimeteric ultramicro method for the determination of free fatty acids. J. Lipid Res. 6: 431-433. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw Hill Book Co., New York. Pp. 109-110. Trout, D. L., E. H. Estes, Jr. and S. J. Friedberg, 1960. Titration of free fatty acids of plasma: a study of new methods and a new modification. J. Lipid Res. 1: 199-202.
NEWS AND NOTES (Continued from page 1133) has its headquarters in Oudenaarde, Belgium. Hubbard Europa operates through subsidiaries in Austria, Belgium, France, Germany, Great Britain, Holland, Ireland, and Italy. The animal health field, including products for poultry breeders, is an important contributor to Merck's growth. Activities in this area represent significant investments of the Company's marketing, production, and research resources. Merck markets products to combat such poultry health problems as coccidiosis and Marek's disease, as well as vitamins and antibiotics to help insure normal health and efficient poultry production. SYMPOSIUM ON LIVESTOCK WASTES The Third International Symposium on Livestock Wastes will be held April 21-24, 1975, at the University of Illinois, Urbana-Champaign, Illinois, U.S.A. The
Symposium is being planned by the American Society of Agricultural Engineers, the professional society that initiated two previous International Livestock Waste Symposia at Michigan State University in 1966, and at Ohio State University in 1971. Eighteen organizations including The Poultry Science Association, Inc., have joined A.S.A.E. in formulating a program that will bring together expertise from a broad spectrum of interrelated fields. These organizations are: American Agricultural Economic Association, American Dairy Science Association, American Institute of Chemical Engineers, American Society of Agronomy, American Society of Animal Science, American Society of Civil Engineers, American Society of Microbiology, Canadian Society of Agricultural Engineering, Farm Equipment Manufacturers Association, Farm and Industrial Equipment Institute, International Commission of Agricultural Engineering, Soil Conservation Society of
(Continued on page 1187)
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male and laying female turkeys. Federation Proc. 32: 933. Bajpayee, D. P., and K. I. Brown, 1972. Effect of photoperiodicity on the circulating levels of estrogens, corticosterone, calcium and free fatty acids in female domestic turkeys (Meleagris gallopavo). Poultry Sci. 51: 1157-1165. Borgstrom, B., 1952. Investigation on lipid separation methods. Separation of cholesterol esters, glycerides and free fatty acids. Acta. Physiologica Scand. 25: 111-119. Dole, V. P., and H. Meinertz, 1960. Microdetermination of long-chain fatty acids in plasma and tissues. J. Biol. Chem. 235: 2595-2599. Eaton, R. P., M. Berman and D. Steinberg, 1969. Kinetic studies of plasma free fatty acid and triglyceride metabolism in man. J. Clin. Invest. 48: 1560-1579. Gilbert, A. B., 1971. The egg; its physical and chemical aspects. In: Physiology and Biochemistry of the Domestic Fowl, ed. by D. J. Bell and B. M. Freeman. Academic Press, New York. Pp. 1379-1399. Heald, P. J., and H. G. Badman, 1963. Lipid metabolism in the laying hen. I. Plasma free fatty acids and the onset of laying in the domestic fowl. Biochim. Biophys. Acta, 70: 381, 388. Heald, P. J., and K. A. Rookledge, 1964. Effect of
R. A. GUILL AND K. W. WASHBURN
E. W. Glazener, 1958. The heritability of body weight, gain, feed consumption and feed conversion in broilers. Poultry Sci. 37: 862-869. Timon, V. M., and E. J. Eisen, 1970. Comparisons of ad libitumand restricted feeding of mice selected and unselected for post-weaning gain. I. Growth, feed consumption and feed efficiency. Genetics, 64: 41-57.
Warwick, E. J., 1958. Fifty years of progress in breeding beef cattle. J. Animal Sci. 17: 922-943. Wilson, S. P., 1969. Genetic aspects of feed efficiency in broilers. Poultry Sci. 48: 487-495. Winters, L. M., and J. McMahon, 1933. Efficiency variation in steers. Minnesota Agr. Exp. Sta. Tech. Bull. 94: 1-28.
W A Y N E L . BACON, MARGERY A . MUSSER AND KEITH I. BROWN
Department of Poultry Science, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691 (Received for publication September 24, 1973)
ABSTRACT Free fatty acid (FFA) concentrations in blood plasma from immature, laying and broody turkey hens were re-examined following removal of phospholipids. A mean value of .21 ± .02 (A. equiv./ml. plasma of FFA was found for immature hens and as they were stimulated into egg production, and no systematic increase was noted. Broody hens had the same concentration as laying and immature hens. Neutral lipid levels (mainly triglyceride) increased from about 3.5 mg./ml. plasma to about 18-25 mg./ml. plasma as the hens reached sexual maturity, then plateaued until a broody period began when they dropped to concentrations comparable to those in immature hens. No correlation was found between FFA and neutral lipid concentrations. Although FFA concentration is much lower than previously reported, 14% of the total daily amount cleared was calculated to be sufficient to account for egg yolk neutral lipid. The remaining 86% would be available for other metabolic functions. POULTRY SCIENCE 53: 1154-1160, 1974
INTRODUCTION
P
LASMA free fatty acid (FFA) levels in chickens have been reported to increase from about 0.3 JJL. equiv. per ml. in non-laying pullets to about 1.0 to 2.0 |JL. equiv. per ml. in laying hens (Heald and Badman, 1963). In this study, FFA levels were determined by the titrimetric method of Dole and Meinertz (1960). A similar study has recently appeared for turkeys (Bajpayee and Brown, 1972). Prior to exposing immature birds to stimulatory lighting, plasma FFA concentration was 1.14 ± .20 |JL. equiv. per ml. After 21 days of stimulatory lighting, FFA concentration had almost doubled to 2.15 ± .11 (ju equiv. per ml. These authors used the method of Kvam
et al. (1964) to determine plasma FFA. This method is based on forming the copper salts of fatty acids (FA) and then colorimetrically determining the amount of copper associated with the fatty acids after they are extracted. The effect of fasting and gonadotrophin injection on plasma FFA levels was studied by Heald and Rookledge (1964). They observed that fasting caused an increase in plasma FFA in normal cocks, but a decrease in plasma FFA in laying hens when initial FFA levels were high and an increase when initial levels were low. Their method of determining FFA was based on the titrimeteric method of Dole and Meinertz (1960). When pituitary powder was injected during four-day starvation of laying hens, plasma FFA levels were maintained, as were plasma total lipids.
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Plasma Free Fatty Acid and Neutral Lipid Concentrations in Immature, Laying and Broody Turkey Hens
PLASMA FATTY ACID AND LIPID
During the course of FFA metabolism studies in laying turkey hens in this laboratory, the concentration of plasma FFA was found to range between .06 to .20 |x. equiv. per ml. In these determinations, PL's were removed by chromatography on silica gel and the FFA's extracted by alkaline ethanol (Borgstrom, 1952) before being quantitated by the method of Novak (1965). This method of FFA quantitation is chemically similar to that of Kvam et al. (1964) except that the cobalt instead of copper salts of the FFA's are formed. Since these results indicated much lower levels of plasma FFA's in laying turkey hens than previously reported, it was decided to do a more complete study of plasma FFA concentration in non-laying and laying turkey hens. In addition, the amount of plasma neutral lipid was determined. MATERIALS AND METHODS Turkeys. Experiment 1. Blood was collected from several laying hens and pooled. Experiment 2. Six young female turkeys
were housed in a floor pen when 24 weeks old, on September 21. They were exposed to natural daylight until February 21, when they were given 14 hours of light per day at about 50 lux intensity. Blood samples were taken about 3 times per week, starting two days before giving stimulatory lighting. About two weeks after full production was attained in all birds, samples were taken at about weekly intervals (see Tables 2 and 4). The birds were moved from the floor pen to cages (48 x 66 x 55 cm.) on March 6, or 3 days before the first egg was laid. Blood Samples. An 8-9 ml. sample of blood was drawn from each bird on each sampling date. The samples were placed in centrifuge tubes containing sufficient heparin to inhibit clotting and the plasmas were collected after centrifugation. The plasmas were then frozen and stored at -20° until analyzed within 2 months. Analyses. Experiment 1. Plasma was pooled from several laying turkeys and 1.0 ml. extracted either with or without added palmitic acid (0.40 9. equiv.) by the method of Trout et al. (1960). Iso-octane was substituted for heptane (Eaton et al., 1969). After extraction, two aliquots with and two without added palmitic acid were set aside for FFA determination by the method of Novak (1965). PL's were removed from the remainder of the extracts by silica gel chromatography (Bio Sil HA, BioRad, Richmond, California) as follows. One and one tenth g. of silica gel were weighed and covered with hexane:diethyl ether (H:D), 1:1. Columns (top 1/2 of a 100 ml. volumetric pipet, i.d. 0.6 cm.) were plugged with glass wool, filled with the silica gel slurry and then allowed to drain dry. The plasma extracts were then added to the columns in 1.0 ml. H:D, 1:1. The sample containers were then washed 2x with additional 1.0ml. amounts of H:D, 1:1 and added to the columns when dry. After this, 7.0 ml.
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If pituitary powder was not injected, both plasma FFA and total lipids levels fell. Neither of the above methods for plasma FFA determination give absolute specificity for FFA concentration, as phospholipids (PL) may interfere if present in either large and/or varying amounts (Dole and Meinertz, 1960; Kvam et al., 1964). It is well known that when a hen enters egg production a lipophosphoprotein complex (LPP) of constant composition can be precipitated from the plasma. LPP is not detected in the plasma of non-laying hens or cocks (Mclndoe, 1959). Furthermore, LPP is composed of 80% lipid, of which about 25% is PL. This indicates large changes in the amounts of PL when non-laying and laying female or when fasting vs. full fed laying female plasmas are compared. If the PL's are not removed prior to FFA determination by the above methods, the levels of circulating FFA's may be overestimated.
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W. L. BACON, M. A. MUSSER AND K. I. BROWN
of Novak (1965). Eighteen to 24 plasma samples were run in each analysis. The standard curve for the FFA's was determined for each analysis with 0, 0.02, 0.04 and 0.06 |x. equiv. of palmitate. To each standard, an aliquot from a blank column was added equivalent to the sample aliquot to compensate for the small amount of background color attributed to column washing by the eluting solvents. FFA concentrations were adjusted to 100 percent recovery. Actual radioactivity recovery was 95 ± 1% (s.e.). RESULTS Experiment 1. It is clearly seen in Table 1 that removal of PL's by the relatively simple Bio Sil HA chromatography step reduces detectable FFA levels by about 75%, while not reducing the recovery of added FFA. After isolation of the FFA's by alkaline ethanol partition, the FFA concentrations for samples with and without added FFA were nearly the same as these respective samples before solvent partition. These data indicate that the high FFA levels previously reported for both laying turkeys (Bajpayee and Brown, 1972) and chickens (Heald and Badman, 1963; Heald and Rookledge, 1964) were probably due to a non-specific overestimation caused by the presence of PL's in their samples. The relatively high concentrations of plasma FFA present in laying in comparison to nonlaying and fasted hens previously reported are probably due to increases in PL con-
TABLE 1.—Free fatty acid concentrations determined on Trout extract, extract after Bio Sil HA chromatography and extract after both chromatography and alkaline ethanol partition
With added FA' Without added FA Difference
'Added 0.40 (x. equiv. palmitic acid. equivalent/ml. plasma ± s.e. Only one sample determined.
2 \x. 3
Trout extract
After Bio Sil chromatography
After chromatography and solvent partition
1.23 ± .042 0.91 ± .04 .32
.68 ± .01 .263 .42
.66 ± .03 .22 ± .01 .44
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of H:D, 1:1 was added to each column and they were allowed to run dry. The tips of the columns were then washed with diethyl ether. After drying and resuspension, an aliquot of each extract was taken for FFA determination. FFA's were extracted from the above purified extracts by partitioning between iso-octane and alkaline ethanol by the method of Borgstrom (1952). An aliquot was then taken in duplicate for FFA determination. Experiment 2. The 1.0 ml. plasma samples were extracted by the method of Trout et al. (1960), using iso-octane instead of heptane and omitting the 0.05% aqueous H 2 S0 4 wash. Since the extracts were to be passed through columns of silica gel the upper layer was completely removed and the lower layer washed 2 x with additional iso-octane and the upper layers combined. These extracts were taken to dryness in a vacuum over at 60° and then chromatographed on Bio Sil HA as in Experiment 1. A tracer dose (6,000 to 7,000 C.P.M.) of 1-C14-Palmitic Acid was added to each sample prior to extraction. The column eluates were collected into tared scintillation vials, the eluting solvents evaporated under vacuum at 60° and the vials then reweighed to determine the amount of neutral lipid, mainly TG and cholesterol, extracted from each sample. After weighing, H:D, 1:1 was added and duplicate aliquots removed to determine both radioactivity recovery and FFA concentration by the method
PLASMA FATTY ACID AND LIPID
1157
TABLE 2.—Free fatty acids and neutral lipids for the various days post stimulatory lighting
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of egg production. The one high mean value at day 14 is probably associated with either the stress of moving the birds from a floor Days FFA ± s.e. Neutral lipids pen to cages or lowered feed consumption u.. equiv./ post 'og.o just prior to initiation of egg production, or lighting ml. plasma mg./ml. ± s.e. mg./m both. Unpublished results from this laborato3.8 .58 ± .06 -2 .15 ± .02 3.3 .52 ± .06 .22 ± .03 0 ry and the report of Meyer et al. (1970) show 4.8 .68 ± .07 .18 ± .02 3 decreased feed consumption at the time of 7.2 .86 ± .04 .19 ± .02 5 10.2 1.01 ± .06 .22 ± .02 7 first egg in turkeys and chickens, respec10.5 1.02 ± .07 .17 ± .01 9 tively. Feed restriction may cause increased 18.5 1.27 ± .04 .17 ± .02 12 14.1 1.15 ± .05 .36 ± .08 14' FFA levels in normal cocks (Heald and 12.2 1.09 ± .06 .29 ± .04 162 Rookledge, 1964) or male turkeys (Bacon and 16.9 1.23 ± .06 .33 ± .08 19 18.7 1.27 ± .07 .24 ± .05 21 Musser, unpublished data). 18.6 1.27 ± .04 .26 ± .07 24 Neutral lipids (mg./ml. plasma) were 17.6 1.24 ± .04 .23 ± .04 26 14.6 1.16 ± .04 .23 ± .05 30 transformed to their log I0 values for analysis 19.3 1.28 ± .05 .14 ± .02 35 of variance, to normalize the variance about 23.7 1.37 ± .04 .17 ± .02 42 26.6 1.42 ± .04 .18 ± .05 49 the means. Table 2 gives both the transformed LSD 3 values ± s.e. and the converted mean values. 26 .22 P < .05 .: ' Birds moved from floor pen to cages on day All comparisons were made using the trans132post lighting. formed values. It is readily apparent that the First eggs were laid from day 16 to 24 post amount of neutral lipids increases during the lighting. 'Method of Tukey (Steel and Torrie, 1960). initial phase after stimulatory lighting, until egg production begins, and then is maintained centration rather than to increases in FFA at this relatively high level as long as the individual hen remains in egg production. concentration. To determine whether the However, a considerable range of levels eiFFA concentration did increase, the second ther between laying hens on a given date experiment was conducted. or within individual laying hens on different dates was found (Table 3). The upper values Experiment 2. The data from 2 days prewere found to be about threefold higher than and the first 49 days post stimulatory lighting the lower values. These data agree with the are given in Table 2. Analyses of variance plasma total lipid data presented by Heald are presented in Table 3. The first eggs from and Badman (1963). all 6 birds were laid from days 16 to 24 (mean 19.0 ± 1.2 days), which was after the birds were moved from the floor pen to cages on TABLE 3.—Analyses of variance of FFA and log, day 13. neutral lipids The FFA level on day 14, or the first day Neutral after moving, was greater (P < .05) than on lipids days - 2 (before lighting) and 35, when all FFA (logio) 1 ms ms Source df of the birds were in full production (Table 2). No significant increase up to time of first Days post .023544* .069786** lighting 16 egg and during initial egg production was .454407** Birds .034460* 5 Residual .014434 80 .010890 noted. This is in contrast to the findings of 1 The mean squares associated with post lighting Heald and Badman (1963) and Bajpayee and and birds were both tested for significance (* = Brown (1972) both of whom reported in- P < .05; ** = P < .01) using the residual mean creases in FFA concentration during initiation squares.
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W. L. BACON, M. A. MUSSER AND K. I. BROWN
TABLE 4.—Free fatty acid and neutral lipids in laying and broody turkey hens Neutral lipids Days post lighting
n
FFA ± s.e. JJL. equiv./ ml. plasma
4 5 4 3 2 3 3
58 61 65 69 75 82 89
2 1 2 3 4 3 3
Broody .41 ± .29 .18 ± .28 ± .18+ .21 ± .16 ±
An overall correlation coefficient with treatment effects absorbed was calculated between FFA and neutral lipid levels. The value of this correlation was -.011 (d.f. = 100) and was not significant (P > .05). This indicates no relationship between plasma FFA and neutral lipid levels as the hens came into and during the first four weeks of production. This is in contrast to the findings of Heald and Badman (1963) and Heald and Rookledge (1964). After 49 days post lighting, broodiness became evident in various hens on various dates. Broodiness is defined as a period in which no eggs are laid for five or more days. Table 4 compares FFA and neutral lipid levels from laying vs. broody hens. It is apparent from this data that there is no sustained difference in FFA level between the laying and broody hens, while neutral lipid levels decrease to levels at or below those present before or on the day of exposing the same birds to stimulatory lighting. The mean value of 0.41 ± .17 p.. equiv./ml. on day 58 in the broody hens was the highest mean observed during the experiment. However, only two hens were broody on this date, one of which assayed at 0.54 \x. equiv./ml. plasma. Individual values up to 0.76 JJU equiv./ml.
hens .01 .02 .06 .01 .01 .04 .02 hens .17 .01 .10 .01 .01 .03
mg./ml.
.02 .02 .05 .10 .02 .03 .09
26.3 21.8 19.8 25.6 30.2 31.4 27.1
.64 ± .01 .45 .58 ± .24 .49 ± .10 .44 ± .06 .37 ± .02 .36 ± .03
4.4 2.8 4.4 3.3 2.8 2.3 2.3
1.42 1.34 1.29 1.38 1.48 1.49 1.41
± ± ± ± ± ± ±
were occasionally noted throughout the experiment. The cause of these high values is unknown and the occurrence was random. DISCUSSION The major finding of the present work is that there is no difference in FFA concentration among immature, laying and broody turkey hens. This is in contrast to the findings of Heald and Badman (1963) and Bajpayee and Brown (1972) for chickens and turkeys, respectively. The increases reported by the above workers can be attributed to the interference of PL in the methods used to determine FFA concentration. In addition, no correlation was found between levels of FFA and neutral lipids in plasma, which is in contrast to the results of Heald and Badman (1963) and Heald and Rookledge (1964) who demonstrated increased levels of FFA coinciding with increased levels of total plasma lipids (neutral lipids + PL's). This correlation can also be ascribed to the lack of specificity in the methods used by the above authors to determine plasma FFA levels. The mean plasma levels of FFA and neutral lipids among the hens while they were laying was .21 ± .02 p.. equiv./ml. and 21.8 ± 1.3 mg./ml., respectively. Plasma neutral lipids
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58 61 65 69 75 82 89
Laying .21 ± .20 ± .26 ± .14 ± .10 ± .16 ± .15 ±
log10 mg./ml. ± s.e.
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PLASMA FATTY A C I D AND LIPID
laboratory (Bacon and Musser) indicate similar turnover times for plasma FFA in laying turkey hens. This value (.14) would indicate the ratio of plasma FFA directly entering plasma LPP neutral lipid TG-FA if LPP neutral lipid TG-FA came directly from plasma FFA and only 5.45 g./day LPP TG-FA is synthesized. Both of these assumptions are highly unlikely. It has been reported recently by Bacon (1973) that plasma neutral lipid TG-FA in laying turkey hens has a turnover time of .43 days instead of 1.1 days. If this value is used to estimate the total amount of plasma neutral lipid removed per day, about 6.4 g. -5- .43 day or 14.8 g./day would be removed. This value is 8.4 g. greater than that calculated from the estimate of 1.1 day for turnover time. This 8.4 g./day is equal to 5.0 mg./min. of LPP TG-FA being removed from the plasma in addition to what is removed by the ovary. This LPP TG-FA could either be removed to the adipose tissue and stored as tissue lipid or re-enter the plasma as FFA. The removal rate of plasma FFA as stearate is 20.4 mg. H- .83 min. or 24.7 mg./min. If we assume that the above 5.0 mg./min. of LPP TG-FA enters the plasma as FFA, then 20% of the plasma FFA could come directly from LPP TG-FA. Calculations such as those above depend upon accurately measuring the concentrations of various plasma constituents and their rates of loss from the plasma and any possible recycling. In the present experiments, we feel that the estimate for plasma FFA has been more accurately made than in previous reports. We are currently studying the kinetics of FFA and LPP TG-FA and hope to report the results of these experiments in the near future. REFERENCES Bacon, W. L., 1973. Turnover time and metabolic clearance rates of very-low-density lipoproteins in
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decreased to 3.3 ± .4 mg./ml. in the broody hens. The difference in the concentration of neutral lipids in the hens when they were laying vs. when they were broody is 18.5 mg./ml. and is probably associated with the LPP complex of Mclndoe (1959). If the plasma volume is assumed to be 4.6% of body weight (McCartney, 1952), which is about 7.5 kg. for this strain, the plasma volume is 345 ml. Plasma content of neutral lipids and FFA is then 6.4 g./hen and 72 JJL. equiv./hen, respectively. If FFA content is multiplied by the molecular weight of stearate (284), an estimate of the plasma FFA content as stearate is obtained. With the present data, this estimate is 20.4 mg./hen. If a hen is laying eggs with 30 g. yolks and each yolk is 33% lipid (Gilbert, 1971), she is producing 10 g. lipid per day as egg yolk. Of this, about 17% is PL, so she is producing about 8.3 g. of neutral lipids per day if she lays at an intensity of 100%. In the present experiment, the six hens laid at an intensity of 70 ± 2%, so their rate of production of neutral lipids would be about 5.8 g./hen/day. When 6.4 g. neutral lipid/hen in the plasma is divided by 5.8 g. neutral lipid/hen/day lost as egg yolk, the value 1.1 days indicates the turnover time in days for the plasma neutral lipids if they are removed only by follicles in rapid development, and exit the bird as egg yolk. If total plasma content of neutral lipids is assumed to be 95% triglycerides (TG) and 90% of the TG is FA, the weight of plasma TG-FA associated with LPP would be 5.45 g. If it is assumed that all of the neutral lipid TG-FA is derived from plasma FFA, and that all plasma FFA enters TG, the plasma FFA would turn over at a rate 5.45 g./l.l day/.0204 g. or 243 times per day, or once each 5.9 minutes. The turnover time of plasma FFA calculated from the data of Heald and Badman (1963) in laying chicken hens is .83 minutes, which is .14 times as large as that calculated above. Unpublished data from this
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gonadal hormones, gonadotrophins, and thyroxine on plasma free fatty acids in the domestic fowl. J. Endocrinol. 30: 115-130. Kvam, D. C , J. G. Schmidt, D. A. Riggilo and D. G. Gallo, 1964. Colorimeteric microdetermination of plasma free fatty acids. J. Pharmaceutical Sci. 53: 988-989. McCartney, M. G., 1952. Total blood and corpuscular volume in turkey hens. Poultry Sci. 31: 184-185. Mclndoe, W. M., 1959. A lipophosphoprotein complex in hen plasma associated with yolk production. Biochem. J. 72: 153-159. Meyer, G. B., S. W. Babcock and M. L. Sunde, 1970. Decreased feed consumption and increased calcium intake associated with pullet's first egg. Poultry Sci. 49: 1164-1171. Novak, M., 1965. Colorimeteric ultramicro method for the determination of free fatty acids. J. Lipid Res. 6: 431-433. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw Hill Book Co., New York. Pp. 109-110. Trout, D. L., E. H. Estes, Jr. and S. J. Friedberg, 1960. Titration of free fatty acids of plasma: a study of new methods and a new modification. J. Lipid Res. 1: 199-202.
NEWS AND NOTES (Continued from page 1133) has its headquarters in Oudenaarde, Belgium. Hubbard Europa operates through subsidiaries in Austria, Belgium, France, Germany, Great Britain, Holland, Ireland, and Italy. The animal health field, including products for poultry breeders, is an important contributor to Merck's growth. Activities in this area represent significant investments of the Company's marketing, production, and research resources. Merck markets products to combat such poultry health problems as coccidiosis and Marek's disease, as well as vitamins and antibiotics to help insure normal health and efficient poultry production. SYMPOSIUM ON LIVESTOCK WASTES The Third International Symposium on Livestock Wastes will be held April 21-24, 1975, at the University of Illinois, Urbana-Champaign, Illinois, U.S.A. The
Symposium is being planned by the American Society of Agricultural Engineers, the professional society that initiated two previous International Livestock Waste Symposia at Michigan State University in 1966, and at Ohio State University in 1971. Eighteen organizations including The Poultry Science Association, Inc., have joined A.S.A.E. in formulating a program that will bring together expertise from a broad spectrum of interrelated fields. These organizations are: American Agricultural Economic Association, American Dairy Science Association, American Institute of Chemical Engineers, American Society of Agronomy, American Society of Animal Science, American Society of Civil Engineers, American Society of Microbiology, Canadian Society of Agricultural Engineering, Farm Equipment Manufacturers Association, Farm and Industrial Equipment Institute, International Commission of Agricultural Engineering, Soil Conservation Society of
(Continued on page 1187)
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male and laying female turkeys. Federation Proc. 32: 933. Bajpayee, D. P., and K. I. Brown, 1972. Effect of photoperiodicity on the circulating levels of estrogens, corticosterone, calcium and free fatty acids in female domestic turkeys (Meleagris gallopavo). Poultry Sci. 51: 1157-1165. Borgstrom, B., 1952. Investigation on lipid separation methods. Separation of cholesterol esters, glycerides and free fatty acids. Acta. Physiologica Scand. 25: 111-119. Dole, V. P., and H. Meinertz, 1960. Microdetermination of long-chain fatty acids in plasma and tissues. J. Biol. Chem. 235: 2595-2599. Eaton, R. P., M. Berman and D. Steinberg, 1969. Kinetic studies of plasma free fatty acid and triglyceride metabolism in man. J. Clin. Invest. 48: 1560-1579. Gilbert, A. B., 1971. The egg; its physical and chemical aspects. In: Physiology and Biochemistry of the Domestic Fowl, ed. by D. J. Bell and B. M. Freeman. Academic Press, New York. Pp. 1379-1399. Heald, P. J., and H. G. Badman, 1963. Lipid metabolism in the laying hen. I. Plasma free fatty acids and the onset of laying in the domestic fowl. Biochim. Biophys. Acta, 70: 381, 388. Heald, P. J., and K. A. Rookledge, 1964. Effect of