Comp. Biochem. PhysioL, 1975, VoL 50B, pp. 237 to 248. Pergamon Press. Printed in Great Britain

THE EFFECT OF TEMPERATURE AND DIET ON THE FATTY ACID COMPOSITION OF JAPANESE QUAIL G. DURAIRAJ* AND ELDEN W. MARTIN Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403, U.S.A. (Received 5 February 1974)

Al~traet--l. Japanese quail were fed 0, 2 or 4% linoleic acid diets from time of hatching and were acclimated to 10, 23 or 42°C. 2. Myristic, palmitic, palmitoleic, stearic, oleic, linoleic, eicosatrienoic and arachidonic constituted more than 95 per cent of fatty acids identified in the carcasses. 3. The ratio of saturated (anoic) to unsaturated (enoic) fatty acids (A/E) was demonstrated for the first time in an avian species to vary in relation to acclimation temperature. 4. The level of unsaturation varied inversely with acclimation temperature. 5. The data indicate that linoleic acid is an essential fatty acid for growth in this species.

INTRODUCTION INVESTIGATIONSwith several of the thermophilic and psychorophilic bacteria have confirmed the concept that an inverse relationship exists between the environmental temperature and the amount of unsaturation of fatty acids found in such organsims (Fulco, 1969; Joyce et aL, 1970; Chan et aL, 1971 ; Ray et aL, 1971 ; Russell 1971). An inverse relationship between the degree of unsaturation of lipids and the mean environmental temperature has been established in several species of animals. Although adaptation and seasonal acclimatization to heat and cold have been studied in birds, very few investigations of biochemical changes have been made with reference to temperature acclimation (Chaffee & Roberts, 1971). Fisher (1962) has analyzed the fatty acids of subcutaneous fat of adult chickens maintained for 6 weeks at 0, 21 and 32°C. He found that there were significantly more unsaturated fatty acids in birds maintained at 0°C than in birds at 32°C, even though the deep body temperature was the same in all birds. He suggested that metabolic adjustments associated with cold adaptation are responsible for changes in body fat composition. Zar (1967) studied the effects of temperature on the fatty acid composition of the brain and muscle of adult house sparrows, Passer domesticus, at four experimental temperatures between - 2 0 and 35°C. The brain fatty acid composition remained constant in all experimental temperatures and the muscle fatty acid profile * Present address: Dept. of Vertebrate Biology (Ford Foundation Program), University of Agricultural Sciences Hebbal, Bangalore 560024 India Outside Asia, address reprint requests to Elden W. Martin.

resembled that found in other birds. However, Barnett (1970) reported that house sparrows were significantly fatter in winter than in summer, and that more of the fatty acids were unsaturated in the winter. Since the results of studies with birds acclimated (short term) in the laboratory (Zar, 1967) compared with those from studies with birds seasonally acclimatized in nature (Barnett, 1970) permit contrasting interpretations, it seemed appropriate to see if total life cycle exposure to selected temperatures might provide data which could resolve this apparent conflict. West & Meng (1968) fed three diets with different fatty acid compositions to captive red polls maintained under constant conditions of temperature and light. They found that the fatty acid composition of depot fat in that species was similar, irrespective of diet. They concluded that environmental conditions of temperature, photoperiod, captivity and the physiological state of the bird (migration, breeding, etc.) exert greater effects on the fatty acid composition of depot fat than diet per se. However, the same investigators (1968) found in studies of willow ptarmigan, Lagopus lagopus, that there was a direct correlation between the dietary fatty acids (C-16 and C-18 series) and those deposited in the birds as total lipids. Only C-24 and C-22 fatty acids which were found in the diet were poorly represented in body fat. Bower & Helms (1969) reported that the relative composition of depot fat in the dark-eyed junco, Junco hyemalis, was neither related to temperature nor to energetic demands, but to dietary intake of various food, which shifted seasonally. Thus a conflict in findings was apparent. Hence, the primary purpose of this investigation was to expose an avian species to a selected range of

237

238

G. DURAIRAJAND ELDEN W. MARTIN

acclimation temperatures to see if exposure to such conditions from hatching to reproductive age would result in a change in the fatty acid composition of the animal. A secondary purpose was to see if the tissue fatty acid composition could be altered by varying the composition of dietary tipids. Japanese quail were chosen as the experimental animals because they require only a short time from hatching to maturity. MATERIALS AND METHODS Eggs of the Michigan strain of Japanese quail, Coturnix coturnix japonica, were incubated at 37°C as per N R C (1969) recommendations. The newly hatched quail were raised in Forma environmental chambers under controlled temperature and humidity conditions. The temperature during the first week was 37°C, the relative humidity 70700 and illumination was constant. Initially, the hatched quail were divided randomly into three groups of fifty-seven each and each group was fed one of the following diets: (a) 070 linoleic acid diet, (b) 2% linoleic acid diet and (c) 4% linoleic acid diet. Food and water were available ad lib. The animals were isolated in individual cages after the first week. Birds that were to be exposed to heat stress were removed to another identical chamber, where the temperature was raised at the rate of 0.5°C/day until it reached 42°C, and this temperature was maintained until the end of the experiment. The remaining animals were exposed to a temperature decrease at the rate of 1°C/day to 23°C. Half of the animals were retained at 23°C and the remainder were transferred to another environmental chamber, in which the temperature continued to be lowered l°C/day until it reached 10°C where it was maintained until the end of the experiment. A photoperiod of 16L and 8D was maintained in all three chambers. The relative humidity was maintained constant in each of the chambers. The diets were designed to be isocaloric and similar in basic content in that they Table 1.

contained the same levels of proteins, minerals, vitamins and antibiotics (Table 1). The diets provided three different levels of linoleic acid as a second variable. Hydrogenated coconut oil was used as an energy supplement in the 0 ~ linoleic acid diet since it contributed none of that specific fatty acid (Walker & Kummerow, 1964). Corn oil was used in the other diets in appropriate quantities to provide the higher levels of linoleic acid. The formulation of this high protein diet was based on the suggestion by Howes (1965) that growing Coturnix quail require 28% protein in a diet which provided an energy content of 2000 productive energy cal/kg. Starch and cellulose were added to the diets in quantities calculated to provide the desired isocaloric content. The levels of calcium and phosphorus present in Fox and Briggs mineral mixture (Table 1) satisfied the recommendations of Krishna & Howes (1967). The diets were prepared by Nutritional Biochemicals Corporation, Cleveland, Ohio. Carcass analysis'

Animals that died during the process of thermal acclimation were frozen and their carcasses were analyzed later. The others were maintained for a minimum period of 2 months, and then sacrificed for lipid analysis. These birds were anesthetized with ether before samples of liver, brain, flight muscles and gonads were quickly removed. The remaining carcass which may be termed as the "rest of carcass" was weighed. All samples were frozen in liquid nitrogen and desiccated under reduced pressures. Lipids were extracted from portions of the same tissues by two techniques: (1) by a mixture of chloroformmethanol (2 : 1), and purified as per the method of Folch et al. (1957); and (2) by Skelly-solve F petroleum ether according to the method No. Aa 4-38 of the American Oil Chemists Society (1963). All vessels used in subsequent operations were flushed with nitrogen to minimize the oxidation of lipids. The extracted lipids were weighed and stored in the presence of "Santoquin" (1,2-dihydro-6-ethoxy-2,2,4-trimethyl quinoline) (Monsanto Co., St. Louis, Mo.) as an antitoxidant.

Composition (per cent of total) of the experimental linoleic acid diets

Ingredient Casein Corn oil Hydrogenated coconut oil Starch Mineral mix1Vitamin mix:~ Choline chloride Procaine penicillin (11 mg/kg) Oxytetracycline (11 mg/kg) Cellulose (Alphacel)

0~o*

Experimental diet designation 2% 4~o

30.90

30'90 3'46

30-90 6-92

7.00 20.67 7-00 0.19 0.20 + + 34.00

28.31 7.00 0-19 0.20 + + 29-94

20-85 7.00 0-19 0.20 + + 33.94

10o.0o%

10o.0o%

100.0o%

* Gram per cent of linoleic acid expected in diet. 5"Mineral and salt mixture (Fox & Briggs, 1960). Vitamin mixture in mg/kg of diet: thiamine HC1, 100; niacin, 100; riboflavin, 16; calcium pantothenate, 20; pyridoxine HCI, 8; biotin, 0-6; folic acid, 4; paraamino benzoic acid, 2.0; menadione, 5.0; alpha tocopheryl acetate, 25; vitamin Ba~, 0.02; vitamin Dz, 600 IUC; vitamin A acetate, 10,000 IUC.

Effect of temperature and diet on fatty acid composition of Japanese quail

Esterification The esterification procedure of Morrison & Smith (1964) was initiated after a known quantity of each lipid sample was dissolved in screw-capped vials. Boron trifluoride (10%) in methanol (Eastman Kodak Co., Rochester, New York) was added at the rate of 2 ml/10 nag of lipid to transesterify the lipids into methyl esters. The vessels containing this mixture were flushed with nitrogen, sealed and heated for 90 min at 70°C, after which they were slowly cooled to room temperature. Two vol. of water were added to each vial and the esters were extracted three times in 3-5 ml of petroleum ether. The total extract was then evaporated to dryness under nitrogen.

Separation of fatty acid methyl esters The method reported by Walker (1969) was used for the separation of fatty acid methyl esters. Each extracted ester was dissolved in 50 ;~ of benzene and applied to a 20 x 20 cm thin layer of silica gel GF 254 (Merck, AG, Darmstadt, W. Germany) as five or six duplicate spots of the same sample. The plates were developed in benzene, dried and sprayed with 2,7-dictdorofluorescein (Eastman Kodak, Rochester, New York). The dried plates were examined under ultraviolet light. The ester bands were scraped off and extracted with 5 nd of petroleum ether (10 min per extraction) at room temperature. The solvent was evaporated under nitrogen and the sample was dissolved in a known volume of carbon-disulfide for gas chromatographic analysis. Gas-liqnid chromatographic analysis was performed with a Barber-Colman Model 5000 gas chromatograph equipped with a hydrogen flame ionization detector and recorder. The samples were injected on an 8 ftx ¼in. copper column packed with 10% SP-1000 on acid-washed 100/120 mesh Chromosorb W (Supelco Inc., Bellefonte, Pa.). After initial conditioning, the column temperature was maintained at 220°C, the injector port temperature at 250°C and the detector temperature at 275°C. Nitrogen was the carrier gas and the flow rate was 50 ml/min. Samples were injected onto the column with 10 tA of carbon-disulfide as the flushing solvent. Each sample was analyzed three times. The peaks obtained on the recorder were identified by comparison of relative retention times with those of known standards under identical operating conditions. The linearity of the detector response was verified by determining known quantities of fatty acid methyl ester standards (Hormel Institute, Minnesota). Reproducibility error was less than 5Yo. Quantification of fatty acids was accomplished by Carrol's (1961) procedure of estimating the relative area under each peak from the values of relative retention time and peak height with suitable modifications as suggested by Brant & Land (1968). Corrections were made for molecular weight of individual acids and changes in instrument sensitivity. Pentadecanoic acid was used as the internal standard and the quantifications were based on peak heights proportional to that acid. The results were expressed in moles per cent (= g moles per cen0. The ratio of saturated (anoic)/unsaturated (enoic) acids, the A/E ratio, was determined. The results were analyzed statistically by using three-way ANOVA (Model II) with repeated measures for unequal sample size (Weiner, 1962). Per cent values were converted to angular transformations using arcsin tables (Rohlf & Sokal, 1969). On finding significant differences between

239

levels of factors, the Student-Newman-Keuls test was used to determine where the differences occurred. Statistical analysis was performed by an IBM 360/50 Computer through the Computational Services Department of Bowling Green State University. RESULTS Myristic, palmitic, palmitoleic, stearic, oleic, linoleic, eicosatrienoic and arachidonic acids constituted more than 95 per cent of the total fatty acids. The brain had a higher per cent of C-22 fatty acids than other tissues. C-16 and C-18 acids were distributed differently in tissues depending on the thermal and dietary influence. The data on fatty acid contents of tissues from quail exposed to different temperatures and diets are available from the authors. These data could not be presented here because they required more space than was available.

Temperature effects The relative quantities of fatty acids in the different tissues showed a general trend toward an increase in unsaturation in cold-acclimated birds and a decrease in unsaturation in heat-acclimated birds. Conversely, birds exposed to higher acclimation temperatures had higher concentrations of saturated fatty acids than those exposed to lower acclimation temperatures (Fig. 1A and B). Birds that were over 63 days old showed a significant response to temperature treatment due to longer exposure in their respective 70 - A

Anoic fatty., acid ---Enoic

faffy acid

Liver

-.,,,,,,// "&

E .,o Li 30

-

f

[

[

I

I

70 - B Rest

40

3oo

Br Rest I

~

I

I

I

io

20

30

40

,50

Temperature, *C Fig. 1. The mole per cent concentration of saturated (anoic) and unsaturated (enoic) fatty acids in Japanese

quail acclimated to three different temperatures. A. Concentration in liver and muscle. B. Concentration in brain and "rest of carcass" fraction.

240

G. DURAIRAJ AND ELDEN W. MARTIN

t e m p e r a t u r e regimes (Table 2). H o t - a c c l i m a t e d birds t h a t were over 63 days old h a d significantly h i g h e r myristic, palmitic a n d stearic acid levels t h a n the cold-acclimated birds. T h e levels o f u n s a t u r a t e d fatty acids such as palmitoleic a n d oleic acids were significantly lower in birds a c c l i m a t e d at 42°C t h a n

in birds a c c l i m a t e d at 23°C. T h e lowest level o f linoleic acid was f o u n d in birds a c c l i m a t e d at 42°C. T h e A / E ratios were calculated for C-16, C-18, C-20 a n d C-22 fatty acids for tissues f r o m m a t u r e b i r d s (Tables 4-7, Figs. 2-4). I n general, the results indicated t h a t the A / E ratio is a f u n c t i o n o f acclima-

Table 2. Mean mole percentages of fatty acids of quail that were older than 63 days arranged in increasing order of composition relative to acclimation temperature (C = cold, N = normal, H = hot) and relative to linoleic acid diet (0, 2 and 4%) Fatty acid

Temperature

14:0

1.8

16:0

C 20"3 C

16:l 18:0 18:1 18:2 18:3 20:0 20:2 20:3 20:4 22:0

2'2 N 23' 1 N 3'8 C 14'8 C 21.0 C 11.8 C 0-2 C 1.8 C 0.9 H 3.6 H 1-1 C 0-7 C

1 '2

H 13'4 N 15"9 H 10.8 H 0.2 N 1.1 N 0.7 N 3"1 N 0.9 H 0.5 N

Diet 4"2 H 26"7 H 3"9 N 25"3 H 27"9 N 18.4 N 0.3 H 3.9 H 1.7 C 5'9 C 1.3 N 1-2 H

1.0

1.3

4% 22.3 2% 2-7 2% 15'0 4% 19-2 0% 8.7 0% 0.2 0% 0.7 -/o~°/ 0.8 2% 1-4 4% 0.8 0% 0.5 2%

2% 24.3 0% 2"8 4% 15"8 2% 19.5 2~o 17.3 2% 0.2 2% 1.0 4% 0.8 4% 3"3 2% 1-2 2% 0.6 4%

6.3 0~, 25.9 4% 3.1 0~; 21 "5 0°4 21.5 4~ 19.1 4% 0.3 4~ 2.9 0~ 2.0 0,~ 5"5 0% 1.3 4% 1.5 0%

Means which share a common underline were not significantly different at the 0"05 level of probability (SNK test). Table 3. A. Mean mole per cent levels of fatty acids in lipids extracted from the experimental diets. Standard errors accompany each mean (calculated from three samples). B. Corresponding per cent levels of the fatty acids available in the diets 0%

2% B

4% B

B

Fatty acid

A Mole %

% available in diet

A Mole %

% available in diet

A Mole %

% available in diet

12:0 14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:1 20:3

46.18+1'23 18.78+0"90 * 13"28 + 0"79 0.37 + 0"02 10.11+0"37 7'59 + 0"24 1.43+0"03 0.07+0-00 0.38+0"01 0.12+0"00 *

3.2326 1'3146

0-21+0"02 0.90+0"57 * 8.83 + 2"02 0.37 + 0.04 3.03+0.73 30.05 + 0-52 53.42+2"31 .0.82+0"15 0.56+0.02 0.49+0-01 0-58 + 0.06

0-0073 0.0311

0-20+0.01 0.60+0.26 0.02+0.00 7-88 + 1-45 0"29 + 0.02 3"76+0"11 29.00_ 1"09 55.17+2.33 0'61 +0.37 0.50+0.06 0-62+0-01 0-59 _ 0.05

0.0138 0.0515 0.0014 0.5453 0.0201 0.2602 2.0068 3-8178 0"0422 0.0346 0-0429 0.0408

* Not detected.

0'9296 0"0259 0.7077 0-5313 0.1001 0.0049 0.0266 0.0084

0.3055 0-0128 0.1048 1.0397 1-8649 0-0284 0.0194 0.0169 0.0201

Effect of temperature and diet on fatty acid composition of Japanese quail Table 4.

241

Mean ratios of anoic/enoic fatty acids in the liver of Japanese quail that were older than 50 days. Anoic/enoic acid ratios of carbon numbers

Diet (linoliec acid 70) Temperature

16

18

20

22

0 0 0

Cold Normal Hot

2.36 +-0"78 2-44+-0.22 37"14+ 9"80

0.46 +-0.07 0.50+-0.05 0"67 +-0'18

0.02 +-0.001 0.04+-0-011 1"22+ 0"840

0.35 +- 0.010 0.49+0.002 --

2 2 2

Cold Normal Hot

4.34 + 0.96 4.14+0.29 39.26+0.05

0.30 +-0-11 0.34+-0.05 0.79+-0.02

0-03 + 0.001 0.05+-0-010 0.17+0.004

0.07 + 0.020 0-29+-0.002 0.12+0.001

4 4 4

Cold Normal Hot

4.30+_1.40 4.36+-0.36 13-45+-1"98

0.35+-0.10 0.37+-0"23 0.69+-0.10

0.04+-0.020 0-29+-0-091 0.14+-0"040

1.29+-0.071 0.18__.0-010 0.69+-0.014

Standard errors are included with the mean values.

Table 5.

Mean ratios of anoic/enoic fatty acids in the muscle of Japanese quail that were older than 50 days Anoic/enoic acid ratios of carbon numbers

Diet (linoleic acid 70) Temperature

16

18

20

22

0 0 0

Cold Normal Hot

4.81 + 1.71 5.83+2.19 6.00 + 1.76

0.72 + 0"01 0.77+0.07 0.82 + 0.20

0.05 + 0-01 0.18+0-11 0.34 + 0.11

0.67 + 0.01 ---

2 2 2

Cold Normal Hot

3.38 + 0.80 3.58+0.81 34.97 + 0.95

0"19 + 0"04 0.29+0.12 1.13 + 0.05

0.09 + 0"02 0.07+0.02 0.05 + 0.05

0'17 + 0-05 0.22+0-05 1.46 + 0.02

4 4 4

Cold Normal Hot

3.54 + 0.09 3.67+0.03 13.85 + 0"31

0.33 + 0.04 0.24+0.01 0.73 + 0-09

0.16 + 0"04 0.12+0.05 0.16 + 0"07

0.51 + 0.05 0.41 +0.01 0.42 + 0-09

Standard errors are included with the mean values.

Table 6.

Mean ratios of anoic/enoic fatty acids in the brain of Japanese quail that were older than 50 days Anoic/enoic acid ratios of carbon numbers

Diet (linoleic acid 70) Temperature

16

18

20

22

0 0 0

Cold Normal Hot

2.66 + 0-83 3.64 + 1.78 6.33 + 2.01

0.82 + 0.01 1.05 + 0.03 1.15 + 0.16

0.13 + 0.03 0.38__+ 0.19 0.60+ 0.18

0.08 + 0.00 ---

2 2 2

Cold Normal Hot

2.43 + 0.26 4.18+0.33 5.49 + 0.05

0-57 + 0.07 0.83+0.05 1.00 + 0-01

0.07 + 0.03 0-09+0.02 0.53 + 0.01

0.02 + 0.00 0.04+0.01 --

4 4 4

Cold Normal Hot

3.07 ___0"63 3.84 + 0.54 6-24+0.12

0.82 + 0"16 0.83 + 0.25 1.45+0.05

0.19 + 0-28 1.00_.+ 0.04 0.10+0.02

0.28 + 0-09 2.23 + 0.89 0.13+0-02

Standard errors are included with the mean values.

G. DURAIRAJAND ELDEN W. MARTIN

242 Table 7.

Mean ratios of anoic/enoic fatty acids in the "rest of carcass" of quail that were older than 50 days

Diet (linoleic acid ~o) Temperature

Anoic/enoic acid ratios of carbon numbers 18 20

16

22

0 0 0

Cold Normal Hot

1"56 ± 0.45 10'60+3"15 8'96+ 1.00

0.67 + 0.31 0'81+0.02 1"14±0.25

0.53 + 0.23 0"89±0.57 1'26±0.63

0.91 ± 0.07 0.42±0.01 3.36± 1.39

2 2 2

Cold Normal Hot

2.58+ 1.42 2-62 + 0.06 6.88 + 0.09

0.31 +0.13 0.21 + 0"10 0.82 + 0.13

0.88+0.12 1-03 + 0-16 3-81 + 1.76

1.11 +0.61 1.55 + 0.65 1.01 ± 0-05

4 4 4

Cold Normal Hot

4-04 + 0.87 13.54+5.51 15.05 + 3.14

0-24 + 0.05 0-36+0.13 0-55 + 0.06

0.62 + 0.23 1.07+0.32 1.11 + 0.34

1-01 + 0.05 1.02+0.29 0-84 + 0.21

Standard errors are included with the mean values. tion temperature. The high A / E values found in hotacclimated birds were characterized by high anoic acid levels and low enoic levels. The low A / E ratio of cold-acclimated birds resulted from a decrease in anoic acid levels with a concomitant increase in 6

116.5

e

1~*15

[]

mE]

e

Rest

4 0

14 []

12 o

8

5_

.

Brain

0 8

6

"5o

4

2

2

T

o

I~3 Cold

oo

t~

Brain

0

IlN0rmal

Muscle

.9

35 []

mNormal

14

BHot

~I 12

Muscle

Liver 4

0

6

42 0

[]

39

37

I1 0

13

[]

Liver 2

Diet, %

,.

Q

0

HNI 4

Fig. 2. Histograms of the ratios of anoic/enoic C-16 fatty acids in liver, muscle, brain and "rest of carcass" of Japanese quail exposed to different experimental temperatures and diets. (Cold, 10°C; normal, 23°C; hot, 42°C.)

0

2

4

Diet, %

Fig. 3. Histograms of the ratios of anoic/enoic C-18 fatty acids in liver, muscle, brain and "rest of carcass" of Japanese quail exposed to different experimental temperatures and diets. (Cold, 10°C; normal, 23°C; hot, 42°C.) enoic'acid levels (Fig. l a and b). Statistical analyses of the A / E ratios of all samples have shown that C-14, C-16, C-18 and C-20 acids were significantly influenced by acclimation temperature, while C-18 and C-20 acids were also influenced by diet (Table 8).

Effect of temperature and diet on fatty acid composition of Japanese quail

243

Table 8. Probability values from statistical comparisons of A/E ratios of C-14, C-16, C-18, C-20 and C°22 acids from the total tissues of quail Fatty acids carbon no. 14 16 18 20 22

Temperature

Diet

0.05 0.05 0-001 0.01 NS

NS NS 0.05 0.05 NS

Temperature x Diet NS NS NS NS NS

NS, Not significant.

Diet effects The diet formulated to contain no linoleic acid (0~) was richer in saturated fatty acids than the other diets (Table 3). Quail fed the 0K linoleic acid diet contained comparatively fewer unsaturated acids than animals fed the other diets. Student Newman Keuls (SNK) tests indicated that the content of myristic acid was significantly higher in animals fed the 0~o diet than those fed the 2 and 4K diets (Table 2). Thus, it is probable that the higher content of 3"81 []

Rest

J

o

myristic acid in coconut oil of the 0K diet accounted for the higher content of that acid in quail tissues. The level of linoleic acid was significantly higher in quail fed the 2 and 4K diets (Table 2). Although thelevels of eicosatrienoic and arachidonic acids were small, the level of eicosatrienoic acid in all birds fed the 0K diet was much higher than that of arachidonic acid. The concentration of arachidonic acid was greater than that of eicosatrienoic acid in the birds fed the 2 and 4K diets. There was a general decrease, in the majority of cases, in the oleic/linoleic ratio in tissues, in response to an increase in the level of linoleic acid in the diet (Figs. 5-7). This decrease in oleic/linoleic acid ratio was seen in all tissues under cold treatment. However, in normal and high temperature environments, the trend to retain linoleic acid was seen in liver, muscle and other body fractions excepting the brain. Many birds fed the 0K diet died within a month. By 5 weeks, 80 per cent of the quail were dead and only 10 per cent of them survived up to 2 months. Only one male survived up to 4 months, and this

7~

j j

o~-

o 0.2

WCold • Normal []Hot

6

5

Muscle

o

u

o 1.22 0"12 ~ 0.08 ~-

Cold(IO"C)

di o

0'17

0 2 9 0"14

i

5 Liver

2

Diet, %

,~

4

Fig. 4. Histograms of the ratios of anoic/enoic C-20 fatty acids in liver, muscle, brain and "rest of carcass" of Japanese quail exposed to different experimental ternperatures and diets. (Cold, 10°C; normal, 23°C, hot, 42°C).

0

2 Diet,

%

Muscle. 4

Fig. 5. Oleic/linoleic (18 : 1/18 : 2) acid ratios in tissues of Japanese quail fed experimental diets at IO°C.

244

G . DURA1RAJ AND ELDEN W . M A R T I N

7"

x

oflinoleic acid feeding (Fig. 8). There was a tendency to incorporate more linoleic acid in eggs depending on the availability of that acid in the diet. Analyses of eggs of the original stock (parental generation) reveal a comparatively low linoleic level.

Normol ( 5

6F

\

5

)

.o

2

4

o

_ -

Shell membrane

[]

Yolk

.__

Muscle

" * ~

[]

O ._c

I O

I

2

o

4

Diet,

c)

%

2

Fig. 6. Oleic/linoleic (18 : 1/18 : 2) acid ratios in tissues of Japanese quail fed experimental diets at 23°C. I 6--

0

5 ~iver

A

8

I I1 C

Source of egg

~4

Fig. 8. Histograms of the oleic/linoleic acid ratios in components of eggs produced by original stock eggs (A) and by quail fed diets which contained 2 (B) and 4% (C) linoleic acid at 23°C.

x

DISCUSSION

Dietary effects

°.°. -

Muscle

0

I

I

2

4

Diet,

%

Fig. 7. Oleic/linoleic (18 : 1/18 : 2) acid ratios in tissues of Japanese quail fed experimental diets at 42°C. occurred under cold acclimation. Those birds fed the 0% diet under heat acclimation showed a leg malady (weak and featherless condition in the tibiotarsal region) and were unable to stand erect. Only those quail fed the 2 and 4% diets, at normal temperature, laid eggs. The size and weight of the eggs produced by birds fed the 2% diet were practically identical with eggs produced by birds on the 4% diet. Two females fed the 2% diet and two females fed the 4% diet laid nine and ten eggs, respectively. The oleic/linoleic acid ratios in yolk and egg membranes showed the effects of the level

While this study was not intended to be a nutritional study in a strict sense, some indication of nutritional requirement has been derived. Linoleic acid has been reported to be an essential fatty acid (EFA) in mammals (Holman, 1964), but its nutritional role in birds is uncertain. The dietary deficiency syndromes for young chickens are suboptimal growth rate, enlarged fatty liver, reduced resistance to respiratory disease, depressed feed consumption as well as reduction in egg laying capacity, fertility and hatchability (Hopkins, 1967). The eicosatrienoic acid concentration increased as a result of that deficiency. Studies in which chickens raised for 25 weeks on a fat free diet, later supplemented with linoleic acid in different doses, revealed that 20 mg was the daily minimum requirement (Menge, 1965a, b, 1968, 1970). A diet containing 2% linoleic acid was required for egg production while 0.75% was the minimum for hatchability. Chickens fed a linoleic acid deficient diet from hatching showed normal fat gain up to 6-8 weeks (Bieri, 1956, 1966).

Effect of temperature and diet on. fatty acid composition of Japanese quail After that stage there was a decrease in weight gain, depigmentation of feathers, scaliness, development of small testes or immature oviducts and increase in water evaporation from the eleventh week. Holman (1964) reported that the ratio of eicosatrienoic/acachidonic acids could serve as an index of EFA deficiency among mammals; a ratio above 0"4 indicated a positive EFA deficiency. In Japanese quail, even though the levels of eicosatrienoic and arachidonic acids were very low, there was an increase in either eicosatrienoic or arachidonic acids in response to increased dietary linoleic acid. The level of eicosatrienoic acid was higher than that of arachidonic acid in all quail fed the 0% diet. However, in quail fed the 2 and 4% diets, arachidonic acid was relatively more abundant than eicosatrienoic acid and resulted in 20:3/20:4 ratios of 0.2 and 0.3, respectively. Thus the quantitative interaction of these two acids in quail generally corresponds to the relationship found in mammals. Since the quantities of arachidonic and eicosatrienoic acids are very small in chickens, Menge (1968) suggested that a oleic/linoleic acid ratio of 4 or more would be a good indication of EFA deficiency. Liver, muscle, brain and "rest of carcass" fractions examined in quail fed the 0% diet showed a very low level of linoleic acid in all tissues except the brain (Figs. 6-8). While the oleic/linoleic acid values for individual tissues do not equal or exceed 5, the "rest of carcass" representing a major part of the bird had a value of 6 in the cold room. Liver had a mean value of 6 under all temperature conditions. The mean ratio in all fractions of birds fed the 0% diet, under all temperature conditions, was 4. Tissues, except brain, from quail fed the 2 and 4% diets had a oleic/linoleic acid ratio below 4 under all temperature conditions. These facts strongly suggest that linoleic acid is an essential fatty acid for quail and that the 0~o diet was insufficient in that acid. It is inferred that the fatty acid composition in brain tissue may not have been greatly influenced by the diet. Birds that survived when fed the 0% diet might have used their parentally derived linoleic acid reserves. Still, most of them died in the early phase of the experiment, probably due to an inadequate level of linoleic acid in their diet. Menge (1967) reported that linoleic acid deficient chickens could be raised for more than 23 weeks but that they exhibited symptoms of an EFA deficiency. Later supplementation corrected the deficiency. This provided some proof of the essentiality of linoleic acid in birds. The poor survival of quail fed the 0% diet provided evidence for the importance of linoleic acid as a dietary constituent in quail. Only one bird fed the 0~o diet survived for 4 months. Fatty acid analysis of the eggs revealed the dietary importance of linoleic acid in reproduction. Both the yolk and the egg membrane linoleic acid contents were higher in quail fed the 4% diet than in those fed

245

the 2% diet (Fig. 8). Our data show that the content of linoleic acid in body tissues was increased as the level of linoleic acid in the diet increased. The results indicate that linoleic acid was an important dietary requirement. This is the first time that this requirement has been clearly demonstrated for this species. The role of linoleic acid in reproduction is worthy of future consideration. It has been suggested that essential fatty acids function as precursors to prostaglandins (Du, 1970). Since dietary linoleic acid controls the tissue levels of eicosatrienoic and arachidonic acids in many animals and because these acids are in turn precursors of PGEt and PGEs, an EFA deficiency may be accompanied by a PGE deficiency. Temperature effects Since temperature acclimation is a multisystem process, this study considered only the interaction between the physical quality of lipids and the physiological activity of tissues. Cells and organisms utilize their ability to vary their composition of fatty acids as a means of maintaining the physical properties of membranes (Dowben, 1969). Even the temperature-induced activation energies of membranous enzymes are considered to be due to the phase transition changes in the lipid part of the membranes (Vagelos et al., 1972). Hence the molecular transformations during thermal acclimation were interpreted to be a physical need for the physiological process. The brain lipids of goldfish show proportionately more unsaturation during cold acclimation than hot acclimation, especially in phosphatidyl choline and phosphatidyl ethanolamine (Roots, 1968). This was also true for phospholipids and neutral lipids of goldfish gill and liver (Storch, 1966), gill mitochondria (Caldwell & Vernberg, 1970) and intestinal mucous membranes (Kemp & Smith, 1970). Similar changes have been reported in hamsters (Fawcett & Lyman, 1954), in adipose tissue (Kodama & Pace, 1963; Williams & Platner, 1967), red blood cell ghosts, liver mitochondria and microsomes (Chaffee et al., 1968). Corresponding changes have been reported in rat mitochondria (Patton & Platner, 1970) and in the liver of Arctic mice (Eyebel & Simons, 1970). Leg bone marrow fatty acids of caribou decrease in saturation in distal regions (Meng et al., 1969). Itoh et al. (1969) have shown that saturated (myristic, palmitic and stearic) acids were present in significantly lower quantities in the extremities of humans exposed to prolonged cold while unsaturated (especially palmitoleic) acids were present in markedly higher concentrations. The relative levels of saturated and unsaturated fatty acids in Japanese quail tissues follow a similar trend with respect to temperature acclimation (Fig. la and b). The ratio of A/E for C-14, C-16, C-18 and C-20 fatty acids increased in quail exposed to higher

246

G. DURAIRAJ AND ELDEN W. MARTIN

acclimation temperature, indicating an inverse relationship between fatty acid saturation and acclimation temperature. This is the first clear demonstration regarding the manipulation of tissue fatty acids by acclimation temperature in an avian species. The poor response shown by house sparrows in Zar's (1967) experiments could be explained in two ways. First, the duration of experimental procedure may not have been sufficient to permit substantial alteration of components at the cellular level. Secondly, the fatty acid profiles of birds caught in nature were probably already programmed, in response to their previous dietary and thermal experience, long before they were subjected to experimental alteration. Miller et al. (1963) reported that pullets, reared for 16 weeks on a practical type diet and subsequently maintained on a low fat diet for 40 weeks, still retained substantial quantities of linoleic and arachidonic acids. This suggests that house sparrows might not have shown changes under relatively short periods of acclimation time. Mepham & Smith (1966) have shown that the transportability of amino acids in the goldfish intestine is directly correlated with acclimation temperature. Studies with artificial membranes using different fatty acids have given similar results (Klein et al., 1970). Significant differences in the mole per cent of myristic, palmitic, palmitoleic, stearic and oleic acids under different acclimation temperatures were demonstrated in Japanese quail (Table 2). As the various proportions of fatty acids in membranes may serve to regulate the appropriate hydrocarbon chain fluidity at the particular environmental temperature, permeability properties may depend upon fatty acid composition. Hence the alteration in fatty acid composition during thermal acclimation could be necessary to keep the hydrocarbon fluidity constant for homeostatic regulation. Also, these changes could permit the organism to enhance its ability to explore a wider range of physical environment. SUMMARY 1. Newly hatched Japanese quail, C. c. japonica, were fed diets containing 0, 2 and 4 ~ linoleic acid, while undergoing acclimation to temperatures of 10, 23 and 42°C under a 16 hr photoperiod. Liver, muscle, brain, gonad and the "rest of carcass" were analysed for per cent lipid and mole per cent levels of fatty acids. 2. Myristic, palmitic, palmitoleic, stearic, oleic, linoleic, eicosatrienoic and arachidonic acids constituted more than 95 per cent of the fatty acids in the total quail. 3. The relative proportion of saturated (anoic) and unsaturated (enoic) fatty acids, as well as the A/E ratio was a function of acclimation temperature. The level of saturation increased after acclimation to higher temperature. Concurrently, the level of

unsaturation increased and saturation decreased with cold acclimation. 4. Five of the major fatty acids (myristic, palmitic, palmitoleic, stearic and oleic) showed significant variations in concentrations under different temperatures of acclimation. The levels of myristic, stearic and palmitic acids were significantly higher in hot-acclimated quail than quail exposed to colder temperatures. The levels of palmitoleic and oleic acids were significantly higher in cold-acclimated quail than in quail acclimated to higher temperatures. The A/E ratios of C-14, C-16, C-18 and C-20 fatty acids were significantly influenced by temperature. 5. This is the first time that it has been clearly demonstrated that the tissue fatty acid composition of an avian species can be manipulated by acclimation temperature. The alteration of fatty acid composition as a response to temperature acclimation is interpreted as a mechanism that functions to keep the physiological properties of membranes fairly constant for homeostasis in spite of varying ambient temperatures. 6. Quail fed a 0 ~ diet which contained hydrogenated coconut oil had a relatively higher content of saturated fatty acids, while the quail fed the 2 and 4 ~ diets which contained corn oil had a higher proportion of unsaturated fatty acids. All fractions of birds fed the 0H diet had significantly higher quantities of myristic acid reflecting the high concentration of this fatty acid in coconut oil. 7. Quail fed the 2 and 4 ~ diets had a significantly higher linoleic acid in their tissues than those fed the 0~o diet. These differences were correlated with the availability of dietary linoleic acid. 8. In quail fed the 0~o diet, eicosatrienoic acid was higher in proportion than arachidonic acid. However, in quail fed the 2 and 4 ~ diets, arachidonic was higher than eicosatrienoic acid. These quantitative relationships of eicosatrienoic and arachidonic acids strongly suggest that linoleic acid is an essential fatty acid in Japanese quail. 9. Examination of oleic/linoleic acid ratios indicated that there was a strong tendency to retain linoleic acid in the tissue depending on the ditetary availability, irrespective of temperature. 10. The high mortality of quail fed the 0 ~ diet and the apparent selective retention of linoleic acid in birds fed the 2 and 4~/o diets strongly support the suggestion that iinoleic acid is an essential fatty acid in Coturnix quail. Acknowledgements--This research was financed in part by funds from the Graduate School, Bowling Green State University, Bowling Green, Ohio. The authors wish to express their sincere gratitude to Dr. T. H. Coleman, Michigan State University, East Lansing, for providing eggs for hatching experimental animals; to Dr. J. Shapiro, Dr. John W. Parrish, Jr. and Mr. Robert Ridgley for help during conduct of the research; and to Dr. George Rendina for his helpful criticisms of the original manuscript.

Effect of temperature and diet on fatty acid composition of Japanese quail REFERENCES

BARNETTL. B. (1970) Seasonal changes in temperature acclimatization of the house sparrow,Passer domesticus. Comp. Biochem. Physiol. 33, 559-578. BIERIJ. G., BRXGGSG. M., SPIVEyFOX, M. R., POLLARD C. J. & ORITZ L. O. (1956) Essential fatty acids in the chick--I. Development of fat deficiency. Prec. Soc. exp. Biol. Med. 93, 237-240. BIERI J. G. & PRIVALE. L. (1966) Linoleic acid requirement of the chick. J. Nutr. 90, 428-432. BOWERE. B. & HELMSC. W. (1969) Seasonal variation in the fatty acids of the slate-colored junco (Junco hyemalis). Physiol. Zool. 41, 157-168. BRANDTA. E. & LANDSW. E. M. (1968) Quantitative gas chromatography, using retention times. Lipids 3, 178-181. CALDWELLR. S. & VERNBERGJ. J. (1970) The influence of acclimation temperature on the lipid composition of fish gill mitochondria. Comp. Biochem. Physiol. 34, 179-191. CARROLL K. K. (1961) Quantitative estimation of peak areas in gas liquid chromatography. Nature, Lend. 191, 377-378. CHAFFEER. R. J., PLATNERW. S., PATTONJ. & JENNYC. (1968) Fatty acids of RBC ghosts, liver mitochondria and microsomes of cold acclimated hamsters. Prec. Soc. exp. Biol. Med. 127, 102-106. CHAFFEER. g. J. & ROBERTSJ. C. (1971) Temperature acclimation in birds and mammals. Ann. Rev. Physiol. 33, 155-202. CHAN M., HIMESR. H. & AKAGIJ. M. (1971) Fatty acid composition of therrnophilic, mesophilic and psychrophilic Clostridia. J. Bact. 196, 876-881. DOWBEN R. M. (Editor) (1969) Biological Membranes. Little, Brown, Boston. Du J. Y. T. (1970) Prostaglandins and essential fatty acid deficiency. Ph.D. dissertation, Ohio State University, Columbus. EYEBELC. E. & SIMONR. G. (1970) Fatty acid composition of the neutral lipids and individual phospholipids of muscle of cold stressed Arctic mice. Lipids 5 590596. FAWCETrD. W. & LYMANC. P. (1954) The effect of low environmental temperature on depot fat in relation to hibernation. J. Physiol. 126, 235-237. FISHER H., HOLLANDSK. G. & WEISSH. S. (1962) Environmental temperature and composition of body fat. Prec. Soc. exp. Biol. Med. 110, 832-833. FOLCH J., LEES M. & SLOANE-STANLEYG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. bioL Chem. 226, 497-509. FULCOA. J. (1969) The biosynthesis of unsaturated fatty acids of Bacilli. J. biol. Chem. 244, 889-895. HOLMAN R. T. (1964) Nutritional and metabolic interrelationships between fatty acids. Fedn. Prec. Fedn. Am. Socs. exp. Biol. 23, 1062-1067. HOPKINSD. T. & NESHEIMW. C. (1967) The linoleic acid requirement of chicks. Poult. Sci. 46, 872-880. HOWES J. R. (1965) Energy, protein, methionine and lysine requirement for growing and laying Coturnix quail. Prec. Southern Agr. Workers 62nd Cony., Dallas, Texas. ITOHS., KUROSHIMAA., DoI K., MORYAK., SHIRATOH. & YOSHIMURAK. (1969) Lipid metabolism in relation to cold adaptability in man. Fedn. Prec. Fedn. Am. Socs. exp. Biol. 28, 960-964.

247

JOYCE G. H., HAMMONDR. K. & Wi-irlx D. C. (1970) Changes in membrane lipid composition in exponentially growing Staphylococcus aureus during the shift 37° to 25°C. J. Bact. 104, 323-330. KEMV P. & SMrra M. W. (1970) Effects of temperature acclimatization on the fatty acids composition of goldfish intestinal lipids. Biochem. J. 117, 9-15. KLEIN R. A., MOORE M. J. & SMrrH M. W. (1970) Diffusion of neutral amino acids across lipid bilayers. J. Physiol., Lend. 210, 33-34. KOOAMAA. M. & PACEN. (1964) Cold dependent changes in tissue fat composition. Fedn. Prec. Fedn. Am. Socs. exp. Biol. 22, 761-765. KRISHNAG. & HOWESJ. R. (1967) Calcium, phosphorus and vitamin D requirements of Coturnix quail, pp. 301-302. Prec. Southern Agr. Workers 64th Cony., New Orleans, LB. MENG M. S., WESTG. C. & IRVINGL. (1969) Fatty acid composition of caribou bone marrow. Comp. Biochem. Physiol. 30, 187-191. MENGE H., CALVERTC. C. & DENTON C. A. (1965a) Further studies of the effect of linoleic acid on reproduction in the hen. J. Nutr. 86, 115-119. MENGE H., CALVERTC. C. & DENTON C. A. (1965b) Influence of dietary oils on reproduction in the hen. J. Nutr. 87, 365-370. MENGEH. (1967) Fatty acid composition and weights of organs for essential fatty acid deficient and nondeficient hens. J. Nutr. 92, 148-152. MENGEH. (1968) Linoleic acid requirement of the hen for reproduction. J. Nutr. 95, 578-582. MENGE H. (1970) Further studies on the linoleic acid requirement of the hen using purified and practicaltype of diets. Poult. Sci. 49, 1027-1030. MEPHAM T. B. & SMITH M. W. (1966) Regulation of amino acid transport across intestines of goldfish acclimatized to different environmental temperatures. J. PhysioL, Lend. 186, 619-631. MILLERE. C., MENGEH. & DENTONC. A. (1963) Effects of long term feeding of fat-free diet to laying hens. J. Nutr. 80, 431-440. MORRISONW. R. & SMITHL. M. (1964) Preparation of fatty acid methyl esters and dimethyl acetals from lipids with boron fluoride-methanol. J. Lipid Res. 5, 600-608. NATIONAL RESEARCH COUNCIL (1969) Coturnix (Coturnix coturnix japonica). Standards and guidelines for the breeding, care and management of laboratory animals. National Academy of Sciences, Washington, D.C. Publication No. 1703. Official and tentative methods of the American Oil Chemists Society (revisions current through 1963). Method No. Aa4-38. PATTON J. F. & PLATNER W. S. (1970) Cold acclimation and thyroxine effects on liver and liver mitochondrial fatty acids. Am. J. Physiol. 218, 1417-1422. RAY P. H., WHITED. C. & BROCKT. D. (1971) Effect of temperature on the fatty acid composition of Thermus aquaticus. J. Bact. 106, 25-30. ROHLF F. J. & SOKALR. R. (1969) Biometry. The Principles and Practice of Statistics in Biological Research. Freeman, San Francisco. ROOTS B. I. (1968) Phospholipids of goldfish (Carassius auratus L.) brain. The influence of environmental temperature. Comp. Biochem. Physiol. 25, 457--466.

248

G. DURAIRAJAND ELDEN W. MARTIN

RUSSELLN. J. (1971) Alteration in fatty acid chainlengths in Microcococcus cryophilus grown at different temperatures. Biochim. biophys. Acta 231, 254-256. STORCH K. S. (1966) Fatty acid composition of lipids extracted from goldfish tissue following temperature acclimation. Ph.D. thesis, University of Illinois, Urbana. WALKER B. L. (1968) Recovery of rat tissue lipids from essential fatty acid deficiency: brain, heart, testes. J. Nutr. 94, 469-474. WALTZERB. L. & KUMMEROWF. A. (1964) Erythrocytes, fatty acid composition and apparent permeability to non-electrolytes. Proc. Soc. exp. Biol. Med. 115, 10991103. WEST G. C. & MENG M. S. (1968) The effect of diet and captivity on the fatty acid composition of red poll (Acanthus flammea) depot fats. Comp. Biochem. Physiol. 25, 535-540.

WEsT G. C. & MENG M. S. (1968a) Seasonal changes in body weight and fat and the relation of fatty acid composition to diet in the willow ptarmigan. Wilson Bull. 80, 426-441. WILLIAMSD. D. d~ PLATNER W. S. (1967) Cold induced changes in fatty acids of the rat and hamster. Am. J, Physiol. 212, 167-172. WINER M. (1962) Statistical Principles in Experimental Design. McGraw-Hill, New York. ZAR J. H. (1967) Temperature induced changes in the fatty acid composition of the brain and muscle of the house sparrow, Passer domesticus. Ph.D. thesis, University of Illinois, Urbana.

Key Word Index--Temperature of acclimation/ adaptation; diet; Japanese quail; Coturnix; fatty acid composition of carcass; linoleic acid diets; anoic/enoic fatty acid ratio; lipids in Coturnix; egg, fatty acid content.