Camp. Biochem. Physiol. Vol. 85A, No. 2, pp. 243-247, 1986 Printed in Great Britain

0300-9629186 $3.00 + 0.00 Pergamon Journals Ltd

EFFECT OF DIETARY 17-P-ESTRADIOL AND 17-a-METHYLTESTOSTERONE ON GROWTH

AND BODY COMPOSITION OF SLOW-GROWING ELVERS (ANGUILLA ANGUILLA L.)

GAD DEGANI MIGAL, Galilee Technological Centre. Kiryat Shmona, 10200, Israel

(Received 16 January 1986)

Abstract-l. Juvenile slow-growing European eels (Anguilla anguillu) receiving a diet supplemented with 1, 5, 10 and 15 mg/kg 17-b-estradiol (ES) and 17-a-methyltestosterone (MT), were grown and studied for one and a half months in fresh water at a temperature of 23°C.

2. At the end of the experimental period the mean weight of juvenile eels (elvers) which received hormones (MT or ES) were higher than the control, however, there was a significant difference (P < 0.05) by r-test) only in elvers receiving 1, 5, 10, 15 mg/kg MT or 15 mg/kg ES in their diet.

3. The growth equations were linear in the elvers receiving MT in the diet and were exponential in the elvers receiving ES in the diet.

4. The percentage of protein retention (PPV), and energy retention (PEV) had negative linear correlations to MT concentration and a positive linear correlation to ES concentrations.

INTRODUCTION

Fish growth can be stimulated by androgens (Mc- Bride and Fagerlund, 1976; Yamazaki, 1976; Degani and Gallagher, 1985). Another important effect of both androgens or estrogens is to produce a monosex population of some fish that may be useful in fish management. Generally, masculinization or femi- nization of teleosts results from treatment with an- drogens or estrogens, respectively (Yumazaki, 1976; Johnstone et al., 1979). Compared to the effect of androgens on fish growth which was studied, little attention has been focused on possible effects of estrogens on fish growth.

Degani and Levanon (1983) characterized the growth of captured European elvers (Anguilla an- guikz) during the initial period of adaptation to artificial food. High mortality rate occurs during this period due to the inability of some elvers to adapt to artificial food. After three months, adapted elvers held at 23°C could be divided into four main groups; fast-growing elvers (3% of total sample), moderately- growing elvers (20% of the total sample), slow- growing elvers (25% of total sample) and non- growing elvers or elvers that lost weight.

The effect of 3,5,3’-triiodo+thyronine (T3) and 17-x-methyltestosterone (MT) on growth and body composition of American eels (A. rostrata) was stud- ied by Degani and Gallagher (1984, 1985).

The present study investigated the differences be- tween the effects of 17-cr-methyltestosterone (MT) and 17-B-estradiol (ES) on body growth com- position, protein retention and energy retention.

MATERIAL AND METHODS

Animals

European glass eels (A. unguillu) caught during their winter migration in February, 1984 in coastal waters off France, were about 7 cm long and weighed about 0.35 g.

Glass eels were placed in indoor containers at a density of 2 kg/m*. They were maintained three months and fed ad libirum. At the end of three months the elvers (juvenile brown eels) which did not learn to eat, lost weight, some of them were dying during the three month growing period. Others, which learned to eat, grew very slowly. and in- creased their body weight during the initial three months only by as much as 1 g (slow-growing eels), these eels were used for this study. The other elvers that grew quite well (Degani and Levanon, 1983) were not used in this study.

Experimental conditions

The slow-growing elvers were placed in nine groups, each group divided randomly into four replicate populations, 20 elvers in each replicate. Each replicate within one of the nine groups received a diet containing 1, 5, 10 or 15 mg/kg of MT, or 1, 5, 10 or 15mg/kg ES. The remaining groups served as an un-treated control. All the elvers were weighed every three weeks on scales with an accuracy of 0.01 g.

The elvers were housed in indoor open containers (40 x 20 x 20 cm) twenty in each. Water flow was 10 ml/hr, compressed air was supplied which provided gentle aeration throughout the experiment. The temperature was main- tained 23 & 1°C and was controlled thermostatically.

Diets

Diet composition is shown in Table 1. The elvers were fed 2.5% body wt (dry wt) twice a day (7 p.m. and 3 a.m.). The food was made once only during the entire experiment from commercially available chicken offal powder and fish meal and frozen at - 20°C. The MT or ES (Sigma) was dissolved in 70% ethyl alcohol (2% of wet wt, by weight).

Body composition

At the end of the experimental period, each group was killed by freezing and, was held at (-20°C) for subsequent analysis body composition. Sample preparation was accord- ing to Boetius and Boetius, 1980, but incorporated a simple modification. The eels were cut into slices about 0.5cm thick and the sample (30g) was homogenized with 30ml of bi-distilled water for 45 set (4500 rpm) in a polytron homogenizer.

In order to determine moisture content, a sample of homogenate was dried overnight in a Hereus Vacuum Oven

243

244 GAD DEGANI

Table 1. Formulation, analysis of the diet

I. Basic ingredient

Fish meal Chicken meal Dairy meal Vitamin premix Gouar Chicken fat Wheat meal Analysis (% dry matter) Protein Lipid Ash Moisture (% aastel

% Feed stuff (as fed)

29 29

5

3; 49.11 14.56 2.18

33.34

at 25°C and <5 mm Hg. After drying, the sample was ground again by pestle and mortar. Fat content was deter- mined after extraction with chloroform and methanol (1:2) under nitrogen, and protein content was determined by the biurate method (Gallagher et al., 1984). Ash was measured by the difference in weight after ignition of sample at 600°C for 5f hr.

Data processing and statistical analyses

The significance of the result was estimated by t-test (Snedecor and Cochran, 1967).

The protein retention (PPV) and PER were calculated according to the equations =

% PPV = Protein gain in fish (dry wt, g)

Protein intake in food (dry wt, g)

% PER = Weight gain (wet wt g)

Protein in food (dry wt g)’

Energy retention (PEV) was calculated as described by National Research Council (NRC)--Warmwater Fish (1977). The percentage of energy retention was calculated according to: I g protein = 5.7 kcal and 1 g fat = 9.5 kcal.

%PEV = Energy gain in fish (kcal)

Energy intake in food (kcai)

RESULTS

I7-oc-Methplfesrosrerone (MT)

The experimental groups which received 1, 5, 10 or 15 mg/kg of MT, grew faster, in weight, than the

WEIGHT OF FISH CO> 15

3 1Ppm

a 5Ppn

125 0 1OPpm

I + ‘5Ppm

Table 2. Growth equations of slow-growing eels from different experimental groups. (P c 0.05 is significant, N.S. no significance)

Concentration Hormone (tig/l) Equation R P

MT I W = 0.0143T + 0.7302 0.99 <0.05 MT 5 W = 0.0119T + 0.7423 0.99 <0.05 MT 10 W = 0.01 L7T + 0.7472 I 10.01 MT 15 W = 0.0112T + 0.7310 0.99 <0.05 Control 0 W = 0.0077T + 0.7206 0.97 NS E I W = 0.7530eoowzT 0.99 co.05 E 5 W = 0.7327e00’“3T I <O.Ol E 10 W = 0.7401e0W9-1T 0.99 co.05 E 15 W = 0.7243e00”‘r 0.99 <0.05 Control 0 W = 0.721 Ie”“s”T 0.98 NS

W is weight of fish (g) and T is the time (days).

control (Fig. 1). At the end of the 45-day feeding period, the mean weights of all the groups receiving MT, were significantly more than the control group (P < 0.01). The group receiving 1 mg/kg MT showed a higher weight gain than the groups that received a higher concentration of MT in their diets, although the difference is significant (P ==z 0.05) only with the group which received 15 mg/kg.

The growth equation was linear in all the experimental groups that received MT in their diet (Table 2).

Percentage of body protein on a dry weight (D.W.) basis was lower in elvers receiving 1 and 5 mg/kg MT in their diet than the elvers receiving 15 mg/kg and the control group, but the differences were not statis- tically significant (Table 3). The percentage of body fat (D.W.) was higher in elvers receiving 1 mg/kg MT in their diet than other experimental groups, and decreased respectively according to the increase in hormone concentration in the diet.

The PPV, PER and PEV percentages were higher in the group that was administered a low concen- tration of the hormone (1 mg/kg) than the group administered a higher concentration of hormone in the diet (5, 10 and 15 mg/kg) (Fig. 3). All the experimental groups which were administered the hormone in the diet had a higher percentage of PPV, PER and PEV than the control (in the control group

1 - v CONTROL

05 3

0 5 10 15 20 25 30 35 40 45 TIME CMYSJ

Fig. 1. Mean weight of elvers fed with different concentrations of 17-a-methyltestosterone.

Effect of hormones on body composition of ekers

Table 3. Body composition of slow growing eels (% dry matter)

Concentration % Protein % Fat % Moisture % Ash

24.5

Hormone

MT MT MT MT

3 40.46 * a.97 5 40.93 f 3.04

30 41.82+0.61 IS 41 .O8 _t 0.34

I 43.45 * 0.86 5 42.S9 +- 0.85

10 42.05 +- 3.83 IS 39.04 * 3.14

(2 zk SD1

33.14+_ 1.22 31.89 + 5.82 27.02 * 0.92 22.13 * 0.45

21.77 to.31 22.74* 1.15

25.48t_O.19 3 1.40 2 0.93

(2 k SD) __....~ 73.59 + 0.3 1 74.05 i: 0.18 74.3 ^+ 0.05

74.55 j, 0.33

74.68 L 0.50 74.71 It: 0.73 75.55 f 0.17 73.42 A 0.17

(% rt SD\

2.22 -+ 0.44 2.36 * 0.29 2.33 + 0.26 1.97+0.10

2.11 + 0.03 2.16 f 0.04 2.10_+0.02 I.98 -+ 0.02

COlltrOI 0 43.59 * 1.16 25.00~ 1.17 74.27 + 0.21 2.32 * 0.05

1.3

1.2

08

07

15 Pm

CONTROL

Fig. 2. Mean weight of elvers fed a diet with different concentrations of 17-fi-estradiol.

50

45

40

35

30

25

20

15

10

5

0

IPW D

/PER b

LPN D

I L t 1 I I i 1 1 I

0 2 4 6 8 10 12 14 16 18 20

HORYONE C~WXNTRATION(U~/ ~0)

Fig. 3. Protein retention (% PPV and % PER) and energy retention (% PEV) of elvers fed a diet with different con~~trations of ~7-ff-methyltestosterone.

% TTY = Protein gain in fish (dry wt g)

Protein intake in food (dry wt g) % PER _ Weight gain in fish (Wet wt g)

- stein in food (Dry wt g)

% PEV = Energy gain in fish (kcal)

Energy intake in food (kcalf’

246 GAD DEGANI

Table 4. The % protein retention (%PPV and %PER) and the % energy retention (%PEV) correlated to hormone concentrations

Treatment hormones Equation R P

MT %PPV= -0.13x + 13.58 0.97 l<O.Ol MT %PER = -0.52.x + 47.05 0.88 co.05 MT %PEV = -0.51.x + 22.84 0.97 <O.Ol

ES %PPV = 0.34.x + I I.71 0.96 <O.Ol ES %PER = 0.48x + 31.10 0.87 <0.05 ES %PEV = 0.34.x + I I .7l 0.96 <O.Ol

x is hormone concentration (mg/kg diet wet weight).

%PPV = 11.74; %PER = 34.15; %PEV = 14.36). A negative linear correlation was found between the hormone concentration and PPV, PER and PEV and there was no effect on pigmentation or eye size (Table 4. Fig. 3).

In each group receiving (ES) in the diet, the mean weight was higher than in the control groups (Fig. 2). At the end of the 45 day feeding period there was a significant difference (P < 0.001) in mean weight between elvers receiving 15 mg/kg in the diet com- pared with the control, such a difference was not found between elvers receiving 1, 5 and 10 mg/kg ES in their diet compared with the control.

The growth equation was exponential in all the experimental groups that received ES (Table 2).

Body composition of elvers at the end of the experimental period is shown in Table 3. The per- centage of protein (DW) in elvers decreased and the percentage of fat (DW) increased with correlation to the increase of the hormone concentration in the diet.

The PPV, PER and PEV, percentages were higher in the groups administered a high hormone concen- tration in the diet, and lower in the experimental groups which received a low dose of hormone in the diet (Fig. 4). In each group receiving ES, a linear correlation was found between %PPV, %PER and %PEV, and the hormone concentration in the diet (Table 4).

DISCUSSION The results of the present study show that both MT

and ES significantly increased the growth of slow- growing European eels (A. anguilla). The growth was more pronounced with low doses of MT than with high doses. However, the growth was more pro- nounced in eels receiving a high dose of ES than a low dose. It seems that both estrogen and androgen might effect growth and that testerone could be synthesized to estradiol. Other differences between the hormones were: the growth equation of eels receiving MT was linear, and the growth equation of eels administered ES was exponential. The meaning of those results are that there was a uniform effect by MT during the experimental period and at the initial period of treatment there was little effect by ES compared to the end of the experimental period.

*The result of the present study substantiated the earlier finding that MT increased growth in low doses, but it became detrimental to growth at a high dosage (Yamazaki, 1976; Lone and Matty, 1980; Degani and Gallagher, 1985). On the basis of the percentage of increase over the control, it appeared that in slow-growing eels a higher dose effected growth, as was found in this study, than on moderate growing eels (Degani, 1985) where 10 and 15 mg/kg MT did not significantly increase the growth over the control. When MT is given in food it increases the growth of different species of Pacific Salmon and Steelhead Trout, while other natural and synthetic anabolic androgenic steroids also have positive effects on fish growth (Hirose and Hibiya, 1968; McBride and Fagerlund, 1976; Yamazaki, 1976; Higgs et nf., 1977; Matty and Cheema, 1978; Lone and Matty, 1980; Degani and Gallagher, 1985).

The result of the present study is in agreement with those studies. Both MT and ES have a positive effect on growth of slow-growing elvers.

Normal growth of fish depends on available food, RNA and protein synthesis (Bullow, 1971; Haines, 1973). The results of the present study show that both MT and ES effect protein retention (PPV) and energy

0 2 4 6 8 lo 12 ‘(4 96 18 20

HORMONE CONCENTMTION bg/Kg)

Fig. 4. Protein retention (X PPV and PER) and energy retention (% PEV) of elvers fed a diet with different concentration of 17-b-estradiol. Legend see Fig. 3.

Effect of hormones on body composition of elvers 247

retention (REV). It seems that both androgenic ster- oids and estrogenic steroids effect protein synthesis and fat. The percentage of total protein and total lipids in fish was effected by steroids that were studied in different fish species. Deposits of fat in muscle in response to steroid feeding has been reported many times in Salamonids (see review of Lone and Matty, 1980). Lone and Matty (1980) found that total lipids increased in Cyprinus carpio by steroid feeding. Sim- ilar results were found in this study, the percentage of lipids (DW) were higher than the control in most of the experimental groups and the percentage was lower than the control.

Finally it has been shown in the present study that MT and ES increase growth of slow-growing elvers and effects protein and fat synthesis. The low dose of MT and high dose of ES have a more significant effect on growth.

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