Corresponding author: Zafer Karslı ( zaferkarsli@hotmail.com ) Academic editor: Sanja Matić-Skoko
© 2021 Zafer Karslı.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Karslı Z (2021) Effects of synthetic androgen (17α-methyltestosterone) and estrogen (17β-estradiol) on growth and skin coloration in emperor red cichlid, Aulonocara nyassae (Actinopterygii: Cichliformes: Cichlidae). Acta Ichthyologica et Piscatoria 51(4): 357-363. https://doi.org/10.3897/aiep.51.70223
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In recent years, the use of anabolic steroids in the coloration and growth of fishes, especially ornamental ones, has attracted great interest. In the ornamental fish industry, it is economically advantageous to produce some species with high commercial value and higher demand, depending on size, color, and sex. Therefore, the most commonly used steroids in this study—i.e., 17α-MT and 17β-Es (E2)—were added to the diet of emperor red cichlid, Aulonocara nyassae Regan, 1922, which has not been previously hormone-treated and has high economic value amongst ornamental fishes. A 60-day study was conducted in a closed system, where the juveniles of the emperor red cichlid were acclimatized with the control/basal diet for 15 days. After which, 15 fish with a similar shade of color and about 5 months old were weighed individually (0.71 ± 0.01 g). All fish were placed into aquaria (30 L) in five different groups, in triplicate. Five different groups consisted of control (without hormone), 50 mg · kg–1 17α-MT, 100 mg · kg–1 17α-MT, 50 mg · kg–1 E2, and 100 mg · kg–1 E2. The fish were fed a diet twice a day (10:00 h, 17:00 h) for 60 days till satiation. During the entire trial period, a 12 h light–12 h dark photoperiod was maintained. Water temperature was measured daily and recorded. Growth parameters of experimental fish were calculated. The color measurement of fish skin (L*, a*, b* values) from around the dorsal section was performed using a colorimeter (Konica Minolta CR 400). Significant differences were determined in the following parameters: weight gain (WG), specific growth rate (SGR), feed conversion rate (FCR), survival rate (SR), condition factor (CF), and sex reversal. The fish group fed with 17α-MT displayed brighter coloration as compared to other groups. Color analysis (instrumental) in terms of L*, a*, and b*, values showed that the group fed with 17α-MT displayed brighter coloration compared to other groups (P < 0.05). In terms of sex reversal, the fish in the 17α-MT groups exhibited 100% masculinization, whereas in E2 supplemented fish groups (50 and 100 mg · kg–1), the feminization rates were 88.88% and 93.33%, respectively. In conclusion, both hormones were found to have positive and negative effects for this fish species, but the 17α-MT hormone was found to be more effective in reversing skin pigmentation, growth, and sexing, which is the main driver in the ornamental fish trade.
coloration, growth performance, ornamental aquaculture, sex reversal
In the recent past, the production of ornamental fish feed was mostly dependent on bycatch and other aquatic organisms being used as live feeds. However, due to the increasing pressure on natural resources, organized industrial sites have been established for the breeding and marketing of ornamental fishes. Nowadays, feed and aquarium equipment used in ornamental fish farming have emerged as an industry that appeals to the global market. In developed and developing countries, the ornamental fish-farming sector has developed with major commercial importance within the aquaculture industry (
Nutrition is one of the most important elements for the sustainability of fish farming. In addition to carbohydrates, proteins, and fats in the structure of the feed used in nutrition, minerals, vitamins, and anabolic steroids are also used as additives.
Anabolic steroids contain androgen (male sex hormone) and estrogen (female sex hormone), which are commonly used as growth supplements in feed. Steroid hormones can be used in feed without any biological loss since they are resistant to catabolism by digestive enzymes. Thus, fish can be supplemented with hormones without incurring any handling stress compared to other invasive methods like injection, etc. Moreover, administering hormones to multiple fish individuals is a simple procedure (
The wide use of growth promoters in animal husbandry using the anabolic sex steroids has attracted their application in the aquaculture industry to shorten the production cycle and lower the production cost (
Anabolic agents (or anabolics) have been defined as substances that enhance the protein synthesis of livestock by positively affecting the nitrogen balance. Therefore, anabolic hormones enable lean meat production and rapid weight gain of fish and livestock with less feed by expediting muscle development. Most of these are steroidal male and female sex hormones (both natural and synthetic) and non-steroidal substances with anabolic effects. Furthermore, growth hormones, somatomedins, insulin, and thyroid gland hormones have also been documented to affect the growth rate positively (
Sex reversal by hormone treatment and its application in certain expensive aquarium fish, especially for which market value depends on the sex, have drawn the attention of many ornamental fish farmers and this situation has demanded the requirement of more studies on this subject. Studies related to color enhancement in ornamental fish farming have gained momentum in recent years and particularly the use of anabolic steroids is of great interest. Apart from sex reversal, steroids are also used for enhancing the growth or color of fish for visual appeal.
Cichlids, being one of the most visually appealing fishes in the industry, are amongst the aquarium fish, for which market value depends on sex. The emperor red cichlid, Aulonocara nyassae Regan, 1922, attracts attention, especially due to its color in shades of red. The colors of the males are much brighter red with a noticeable difference compared to the females and their dorsal and anal fins are more pointed. Females also usually have grey and dark tones. In this study, the emperor red cichlid was used due to its importance in the aquarium industry and there is a lack of information on the effect of sex steroids on the growth and coloration of this species.
Experimental design. A 60-day study was conducted in a closed system, where the juveniles of emperor red cichlid (A. nyassae) were acclimatized with the control/basal diet for 15 days. After which, 15 fish with a similar shade of color and about 5 months old (unsexed) were weighed individually (0.71 ± 0.01 g) and placed into each aquarium (30 L) in five different groups, in triplicate. Five different groups consisted of control (without hormone), 50 mg · kg–1 17α-MT, 100 mg · kg–1 17α-MT, 50 mg · kg–1 17β-Es (E2), and 100 mg · kg–1 17β-Es (E2). The basal feed had the approximate composition of 46.5% crude protein, 8.2% crude fat, 3.3% crude fiber, 5.0% moisture, and 5.3% crude ash. The fish were fed twice a day (10:00 h, 17:00 h) for 60 days a diet till satiation. Experimental fish were weighed at the beginning of the experiment and the last day of the experiment (on the 60th day). Waste materials were siphoned out every day and replacement water was added. In each experimental treatment group, fish were acclimatized to aquarium and feeding conditions for a week. The temperature of the aquarium unit was set to have a water temperature of 24 ± 1°C throughout the experiment by a room air conditioner. Aquaria were aerated using an air stone.
Hormones were added to the commercial diet and the proximate composition of the feed is provided in Table
During the entire trial period, a 12 h light–12 h dark photoperiod was maintained. Water temperature was measured daily and recorded as 26 ± 1°C, while pH, dissolved oxygen, and NH4+ values were evaluated weekly and maintained at 6.5–8.5, > 5 mg · L–1 and 2–5 mg · L–1, respectively.
Proximate composition of the experimental diets used for feeding emperor red cichlid, Aulonocara nyassae.
Chemical composition | Diet group | ||||
---|---|---|---|---|---|
Control | 50 mg 17α-MT | 100 mg 17α-MT | 50 mg E2 | 100 mg E2 | |
Crude protein [%] | 46.5 | 46.5 | 46.5 | 46.5 | 46.5 |
Crude lipid [%] | 8.2 | 8.2 | 8.2 | 8.2 | 8.2 |
Crude fiber [%] | 3.3 | 3.3 | 3.3 | 3.3 | 3.3 |
Moisture [%] | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 |
Ash [%] | 5.3 | 5.3 | 5.3 | 5.3 | 5.3 |
NFE | 31.7 | 31.7 | 31.7 | 31.7 | 31.7 |
17α-Methyltestosterone [mg · kg–1] | — | 50 | 100 | — | — |
17β-Estradiol [mg · kg–1] | — | — | — | 50 | 100 |
Proximate analysis | |||||
GE, [kj · g–1] | 19.69 | 19.69 | 19.69 | 19.69 | 19.69 |
Color measurements. The color measurement of fish skin (L*, a*, b* values) (
The hue (H°ab), also calculated with a* and b* values, is an angle value representing the shade of redness, yellowness, greenness, and blueness of the skin color. Hue degree indicates red as it approaches 0°, yellow as it approaches 90°, green as it approaches 180°, and blue as it approaches 270°.
ΔE is the color difference value and was calculated with L*, a*, and b* measurements obtained at the beginning and the end of the trial (
C * = (a*2 + b*2)0.5
H°ab = 180 + tan–1 (b* · a*–1) for a* < 0
H°ab = tan–1 (b* · a*–1) for a* > 0
ΔE = [(Lt − L0)2 + (at − a0)2 + (bt − b0)2]0.5
Where Lt is lightness at the end of the experiment, L0 is lightness in the initial of the experiment; at is red to green degree at the end of the experiment, a0 is red to green degree in the initial of the experiment; bt is yellow to blue degree at the end of the experiment, b0 is yellow to blue degree in the initial of the experiment.
Growth parameters. Growth parameters of experimental fish were calculated with the following equations:
The specific growth rate (SGR) [%· day−1], the weight gain (WG) [%], the survival rate (S) [%], the Feed Conversion Ratio (FCR), and Fulton’s condition factor (K) were calculated using the respective formulas:
SGR = 100 [ln WF – ln W0] · t–1
W G =100 (WF – W0) · W0–1
S = 100 (NF – N0)
FCR = WF · WG–1
K = 100(WW · LT–3)
where W0 and WF are the initial and final body weight and t is the time (feeding period) in days; Nt is the number of fish at the end of experiment and N0 is the number of fish at the beginning of the experiment); WF is the weight [g] of the food given and LT is the total length [cm] of the fish.
Sex parameters. In the experiment, the sex reversal of the fish was determined by looking at the primary and secondary sex characteristics.
Statistical analyses. Statistical analyses were conducted with “Minitab Release 17 for Windows” software. Data were subjected to one-way analyses of variance (ANOVA), followed by the Tukey test at a significance level of 5% (0.05).
Ethical note. This research was approved by the Ethics Committee of Sinop University with Reference No. MYO-1901-16-22. All the procedures applied in this study took into account the importance of preventing or at least minimizing, any kind of animal discomfort or suffering.
At the end of the 60-day feeding period, it was observed that the fish group fed a higher dose of 17α-MT (100 mg · kg–1) had a significantly positive effect (P < 0.05) in terms of weight increase, weight gain, FCR, and SGR. Growth parameters in the group fed 17α-MT (50 mg · kg–1) were also better compared to the group without 17α-MT. However, no change was observed in the fish groups fed E2. The highest survival rate was observed in the control group (93.33 ± 2.45%) followed by 50 mg · kg–1 17α-MT treated group (86.67 ± 7.35%) (P < 0.05). The survival rates of the remaining fish groups (50–100 mg · kg–1 E2 and 100 mg · kg–1 17α-MT) were low.
In terms of sex reversal, the fish in 17α-MT groups exhibited 100% masculinization, whereas in E2 supplemented fish groups (50 and 100 mg · kg–1), the feminization rates were 88.88% and 93.33%, respectively (Table
Growth performance, feed conversion ratio (FCR), survival, conditions and sex ratio of experimental feeds fed for 60 days to emperor red cichlid, Aulonocara nyassae.
Parameter | Control | 50 mg 17α-MT | 100 mg 17α-MT | 50 mg E2 | 100 mg E2 |
---|---|---|---|---|---|
Initial weight [g] | 0.71 ± 0.01a | 0.71 ± 0.02a | 0.71 ± 0.01a | 0.71 ± 0.01a | 0.71 ± 0.02a |
Final weight [g] | 1.54 ± 0.13c | 1.92 ± 0.17b | 2.23 ± 0.19a | 1.62 ± 0.14c | 1.59 ± 0.11c |
Weight increase [g] | 0.83 ± 0.09c | 1.21 ± 0.13b | 1.52 ± 0.11a | 0.91 ± 0.12c | 0.88 ± 0.07c |
Weight gain [g] | 116.90 ± 13.4c | 170.42 ± 12.09b | 214.08 ± 18.4a | 128.17 ± 15.07c | 123.94 ± 14.3c |
SGR | 1.29 ± 0.18c | 1.66 ± 0.19b | 1.91 ± 0.26a | 1.37 ± 0.16c | 1.34 ± 0.15c |
FCR | 3.23 ± 0.71a | 2.32 ± 0.43c | 2.19 ± 0.31c | 2.93 ± 0.66b | 3.01 ± 0.53b |
K | 1.11 ± 0.12b | 1.23 ± 0.15a | 1.09 ± 0.08b | 1.24 ± 0.13a | 1.27 ± 0.07a |
Survival rate [%] | 93.33 ± 2.45a | 86.67 ± 7.35a | 73.33 ± 9.24b | 68.8 ± 11.21bc | 62.20 ± 12.4c |
Males [%] | 28.89b | 100a | 100a | 11.11c | 6.67c |
Female [%] | 71.11b | 0c | 0c | 88.88a | 93.33a |
Color analysis (instrumental) in terms of L*, a*, and b* values showed that the group fed 17α-MT displayed brighter coloration compared to other groups (P < 0.05). In terms of Hue (H°ab) values, the skin color of fish in 17α-MT supplemented groups showed more intense shades of red, while the skin colors of fish in E2 supplemented groups were observed to be shades of grey-brown closer to the shade of yellow color. The maximal change in color difference (ΔE) was observed in fish groups supplemented with 50–100 mg · kg–1 17α-MT (Table
Color parameters measured in fish skin of juvenile emperor red cichlid (Aulonocara nyassae) during the experimental period.
Group | |||||
---|---|---|---|---|---|
Color parameters and periods | Control | 50 mg 17α-MT | 100 mg 17α-MT | 50 mg E2 | 100 mg E2 |
L * | |||||
Beginning of experiments | 41.57 ± 0.11 | 41.57 ± 0.11 | 41.57 ± 0.11 | 41.57 ± 0.11 | 41.57 ± 0.11 |
30th day | 40.62 ± 0.13 | 37.60 ± 0.09 | 29.13 ± 0.05 | 47.65 ± 0.12 | 47.28 ± 0.08 |
60th day | 40.15 ± 0.07b | 26.11 ± 0.18c | 17.22 ± 0.24d | 54.34 ± 0.32a | 53.16 ± 0.29a |
a * | |||||
Beginning of experiments | –2.11 ± 0.04 | –2.11 ± 0.04 | –2.11 ± 0.04 | –2.11 ± 0.04 | –2.11 ± 0.04 |
30th day | –1.57 ± 0.08 | 7.89 ± 0.11 | 8.23 ± 0.09 | –1.67 ± 0.14 | –1.44 ± 0.17 |
60th day | 2.06 ± 0.03b | 8.17 ± 0.21a | 9.86 ± 0.15a | –1.28 ± 0.04c | –1.53 ± 0.06c |
b * | |||||
Beginning of experiments | 4.82 ± 0.09 | 4.82 ± 0.09 | 4.82 ± 0.09 | 4.82 ± 0.09 | 4.82 ± 0.09 |
30th day | 15.21 ± 0.24 | 9.74 ± 0.15 | 8.48 ± 0.05 | 4.89 ± 0.12 | 4.14 ± 0.16 |
60th day | 9.78 ± 0.06a | 5.12 ± 0.13b | 4.09 ± 0.09b | 5.36 ± 0.14b | 5.29 ± 0.19b |
H°ab | |||||
Beginning of experiments | 113.64 ± 0.14 | 113.64 ± 0.14 | 113.64 ± 0.14 | 113.64 ± 0.14 | 113.64 ± 0.14 |
30th day | 95.89 ± 0.19 | 50.99 ± 0.11 | 45.86 ± 0.23 | 108.86 ± 1.36 | 109.79 ± 1.44 |
60th day | 78.11 ± 0.27b | 32.07 ± 0.48c | 22.53 ± 0.32c | 103.43 ± 2.14a | 106.13 ± 1.56a |
C * | |||||
Beginning of experiments | 5.26 ± 0.08 | 5.26 ± 0.08 | 5.26 ± 0.08 | 5.26 ± 0.08 | 5.26 ± 0.08 |
30th day | 15.29 ± 0.45 | 12.53 ± 0.37 | 11.82 ± 0.33 | 5.17 ± 0.12 | 4.38 ± 0.15 |
60th day | 9.99 ± 0.02a | 9.64 ± 0.24a | 10.67 ± 0.21a | 5.51 ± 0.01b | 5.51 ± 0.03b |
ΔE | |||||
0th–60th day | 6.63 ± 0.35d | 18.57 ± 0.44b | 27.14 ± 0.41a | 12.81 ± 0.23c | 11.61 ± 0.29c |
In the ornamental fish industry, it is economically advantageous to produce certain species that have high commercial value and more demand depending on size, color, and sex. Therefore, in this study, the most commonly used steroids (namely, 17α-MT, and E2) were supplemented to the feed of emperor red cichlid.
Fish groups fed 17α-MT hormone had a better growth rate. These results are in agreement with the studies on Labidochromis caeruleus (see
In the presently reported study, fish groups fed E2 had no significant changes in growth parameters, which is in agreement with the results studied on Amatitlania nigrofasciata (see
Apart from beneficial effects, the most pronounced negative effect of hormone supplementation is on the survival rate, which, in turn, is dose-dependent, as well as species and sex specific. In this study, the survival rate was found to be high in the control group and the fish group supplemented with the low dose (50 mg · kg–1) of 17α-MT. On the other hand, the survival rate was low in all fish groups supplemented with E2 and the fish group treated with a high 17α-MT ratio. It has been reported that the high concentration of 17α-MT and E2 hormones lower the survival rate of swordtail (
When the sex ratios were examined, it was determined that the masculinization rate was 100% in fish groups with 17α-MT hormone. The groups supplemented with E2 had a positive impact on feminization compared to the control group, although it was not as effective as the 17α-MT in masculinization.
The visual appeal and color of aquarium fish are important criteria for their marketability. In the presently reported study, enhancement in coloration of fish caused by hormone treatment was statistically demonstrated with the applied method and supported by the results of analyses. According to color analyses conducted at the beginning of the study, hue (H°ab) values indicated shades of grey-brown as the fish were at the early developmental stage. The color differences amongst groups were apparent according to the analyses carried out on day 30. At the end of the study, the desired coloration was determined in 17α-MT-supplemented fish groups and there were differences between these groups over control. However, no such significant improvement in coloration was seen in the groups supplemented with E2. As male individuals of emperor red cichlid appear as bright red, the primary reason for enhanced coloration in fish groups supplemented with 17α-MT hormone may be attributed to efficient masculinization in these groups, as it caused production of 100% male population.
In the presently reported study, the juveniles of emperor red cichlid (A. nyassae) which were supplemented with 100 mg · kg–1 17α-MT displayed the highest growth and weight gain. On the other hand, group E2 was found to be effective in sex reversal, similar to group 17α-MT. When the results of growth, coloration, sex reversal, and survival rate parameters are analyzed in this study, they were found to be important in the ease of marketing in the ornamental fish trade. Especially, skin coloration is the leading factor that determines the market value of ornamental fish. The discoloration and faded color in aquarium fish cause adverse effects on marketing. Aquarium fish cannot synthesize color pigments sufficiently in the culture environment and thus supplementation of synthetic or natural color through feed should be provided. When the results of growth, coloration, sex reversal, and particularly survival rate are evaluated, the most effective hormone dose for emperor red cichlid fish was determined to be 17α-MT at 50 mg · kg–1.
The author is grateful to Assist. sDr Dilek Şahin and Assist. Dr Meryem Öz for contributing to this research.