Unveiling the Effects of Fennel (Foeniculum vulgare) Seed Essential Oil as a Diet Supplement on the Biochemical Parameters and Reproductive Function in Female Common Carps (Cyprinus carpio)

Abstract

The present study aimed to investigate the effect of Foeniculum vulgare essential oil on the health of the common carp (Cyprinus carpio). A total of 120 healthy fish were provided with feed containing 200, 400, and 600 mg/kg of F. vulgare oil for 60 days. Findings revealed that the oral administration of 200 mg/kg of F. vulgare oil significantly increased final weight, weight gain, and specific growth rate compared to the control group (p < 0.05). Plasma biochemical enzyme levels remained unchanged in 200 and 400 mg/kg treatments, whereas they were found to be significantly increased in treatments with 600 mg/kg. Although no significant alterations in glucose, triglyceride, and cholesterol levels were observed, the treated groups exhibited significant increases in total protein, albumin, globulin, and creatinine levels. Results also indicated significantly decreasing levels of glutathione peroxidase, whereas superoxide dismutase activity increased. The gonadosomatic index showed notable improvement in the 200 and 400 mg/kg groups. Furthermore, plasma concentrations of estradiol and testosterone were significantly affected by doses of 400 and 200 mg/kg. Findings suggest that, following the administration of F. vulgare extract, the reproductive and general health of the fish appears to be improved. Nevertheless, it is recommended to supplement fish diets with up to 200 mg/kg of F. vulgare extract to improve their reproductive and general health. Concentrations above this limit can potentially cause harm.

Full text

Citation: Ahmadniaye Motlagh, H.;
Horie, Y.; Rashid, H.; Banaee, M.;
Multisanti, C.R.; Faggio, C. Unveiling
the Effects of Fennel (Foeniculum
vulgare) Seed Essential Oil as a Diet
Supplement on the Biochemical
Parameters and Reproductive
Function in Female Common Carps
(Cyprinus carpio). Water 2023,15, 2978.
https://doi.org/10.3390/w15162978
Academic Editor: Dapeng Li
Received: 6 July 2023
Revised: 11 August 2023
Accepted: 15 August 2023
Published: 18 August 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
water
Article
Unveiling the Effects of Fennel (Foeniculum vulgare) Seed
Essential Oil as a Diet Supplement on the Biochemical
Parameters and Reproductive Function in Female Common
Carps (Cyprinus carpio)
Hamidreza Ahmadniaye Motlagh 1, * , Yoshifumi Horie 2, Hediye Rashid 1, Mahdi Banaee 3,*,
Cristiana Roberta Multisanti 4and Caterina Faggio 4, *
1Department of Fisheries, Faculty of Natural Resources and Environment, Ferdowsi University of Mashhad,
Mashhad 9177948974, Iran; rashid.hediye@gmail.com
2Research Center for Inland Seas (KURCIS), Kobe University, Fukaeminami-Machi, Higashinada-ku,
Kobe 658-0022, Japan
3Aquaculture Department, Faculty of Natural Resources and the Environment, Behbahan Khatam Alanbia
University of Technology, Behbahan 6361663973, Iran
4Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina,
98166 Messina, Italy
*Correspondence: ahmadnia@um.ac.ir (H.A.M.); mahdibanaee2@gmail.com (M.B.); cfaggio@unime.it (C.F.)
Abstract:
The present study aimed to investigate the effect of Foeniculum vulgare essential oil on the
health of the common carp (Cyprinus carpio). A total of 120 healthy fish were provided with feed
containing 200, 400, and 600 mg/kg of F. vulgare oil for 60 days. Findings revealed that the oral
administration of 200 mg/kg of F. vulgare oil significantly increased final weight, weight gain, and
specific growth rate compared to the control group (p< 0.05). Plasma biochemical enzyme levels
remained unchanged in 200 and 400 mg/kg treatments, whereas they were found to be significantly
increased in treatments with 600 mg/kg. Although no significant alterations in glucose, triglyceride,
and cholesterol levels were observed, the treated groups exhibited significant increases in total protein,
albumin, globulin, and creatinine levels. Results also indicated significantly decreasing levels of
glutathione peroxidase, whereas superoxide dismutase activity increased. The gonadosomatic index
showed notable improvement in the 200 and 400 mg/kg groups. Furthermore, plasma concentrations
of estradiol and testosterone were significantly affected by doses of 400 and 200 mg/kg. Findings
suggest that, following the administration of F. vulgare extract, the reproductive and general health of
the fish appears to be improved. Nevertheless, it is recommended to supplement fish diets with up to
200 mg/kg of F. vulgare extract to improve their reproductive and general health. Concentrations
above this limit can potentially cause harm.
Keywords: Cyprinus carpio; fennel extract; phytoestrogen; reproduction
1. Introduction
Inappropriate environmental conditions in captivity can impede the normal reproduc-
tive abilities of numerous aquatic species [
1
]. This is primarily attributed to the absence of
environmental cues necessary for initiating the reproductive process in breeding conditions.
In this context, natural or synthetic hormones are commonly used to overcome this issue
and produce high-quality gametes [2].
Studies showed that aquatic animals’ reproductive systems and other organs are sus-
ceptible to damage due to the prolonged presence of hormones. According to Lange et al. [
3
],
natural and synthetic steroids have differing effects on ovarian tissue, growth, and matura-
tion. Synthetic steroids tend to have greater efficacy due to their resistance to degradation
during digestion, which slows down their excretion compared to natural hormones [
2
,
4
].
Water 2023,15, 2978. https://doi.org/10.3390/w15162978 https://www.mdpi.com/journal/water

Water 2023,15, 2978 2 of 13
Conversely, several plant-based compounds have been studied for their protective and
performance-enhancing effects on fishes [
5
]. For instance, plant-based steroid compounds
in biological equilibrium do not accumulate in the body like synthetic steroids and therefore
do not cause harmful effects [
6
]. This is why plants containing phytoestrogens are a better
alternative to industrial estrogens, as they offer a safer option [
7
]. Phytoestrogens are
plant-derived compounds that have a similar structure to the hormone estrogen found in
humans and animals. Phytoestrogens can be found in various plant-based foods, including
soybeans, flaxseeds, chickpeas, lentils, and fennel [
8
,
9
]. Phytoestrogens can exert weak
estrogenic or anti-estrogenic effects in the body, depending on factors such as the type of
phytoestrogen and the amount consumed. They can bind to estrogen receptors in the body
and mimic some of the actions of estrogen. Some studies have shown that they can help bal-
ance, regulate, and increase levels of the luteinizing hormone (LH) and follicle-stimulating
hormone (FSH), which are important hormones for ovulation [10].
There is some evidence to suggest that phytoestrogens may have a positive effect
on reproductive health in female fishes, including their potential to induce ovulation.
Phytoestrogens can have diverse effects on female fishes, depending on the species, the
dose, and other factors. In some cases, phytoestrogens may benefit a female fish’s health.
However, exposure to high levels of phytoestrogens or prolonged exposure over time
can disrupt the normal endocrine function in a female fish, leading to adverse effects
such as impaired fertility, altered fecundity, and increased risk of ovarian degeneration.
Furthermore, because phytoestrogens are structurally similar to the hormone estrogen,
they can interfere with the action of natural estrogen in the body, potentially leading to
hormonal imbalances and related health issues [
11
]. Therefore, more research is needed to
understand this relationship fully.
In certain fish species, phytoestrogens have been reported to stimulate the production
of endogenous estrogens, which can help induce ovulation. Studies showed that feeding
a female African catfish a diet containing a soybean meal rich in phytoestrogens resulted
in higher levels of endogenous estrogens and increased ovulation rates [
12
]. However,
the effects of phytoestrogens on fish reproduction can be complex and depend on various
factors, such as the species of fish, the dose and duration of exposure to the phytoestrogen,
and the timing of administration concerning the reproductive cycle.
Fennel (Foeniculum vulgare) contains phytoestrogens, plant compounds that mimic
the hormone estrogen in the body. F. vulgare contains a variety of bioactive compounds,
including phenolic compounds (flavonoids, phenolic acids, and tannins), essential oils
(anethole, fenchone, and estragole), terpenes (limonene, pinene, and myrcene), coumarins
(scopoletin and umbelliferone), and alkaloids (anabasine and nicotine) [
13
]. The primary
phytoestrogen found in fennel is called anethole. Anethole is believed to have weak
estrogenic activity, meaning it can bind to estrogen receptors in the body and exert some
estrogen-like effects [
8
]; however, the extent of these effects is relatively mild compared to
those of actual estrogen.
Fennel has undergone extensive research revealing its numerous benefits, which
encompass antimicrobial, antifungal, anti-inflammatory, antioxidant, anxiolytic, cardio-
protective, and potential hormonal properties [
13
]. Notably, fennel acetone extract shows
promise in menstrual cycle regulation [
14
], and it may affect hormone levels by increasing
the follicle-stimulating hormone while decreasing the luteinizing hormone and testos-
terone [
15
]. Moreover, fennel essential oils notably influence oocyte maturation and sexual
maturity in fishes [16,17].
Although phytoestrogens such as F. vulgare may potentially induce ovulation in some
fish species, further research is needed to fully understand their effects and how they could
be used in aquaculture or other applications. This study primarily aims, for the first time, to
examine the impact of fennel essential oil on the growth performance, reproductive indices,
and liver enzymes of the female common carp (Cyprinus carpio), as it ranks as the fourth
most economically valuable aquaculture species.

Water 2023,15, 2978 3 of 13
2. Materials and Methods
2.1. Preparing Fennel Essential Oil and Determining the Amount of Effective Substance
In this study, the fennel essence was extracted by mixing 100 g of washed and dried
fresh fennel seeds with 1000 milliliters of distilled water in a Clevenger apparatus for
four hours. The resulting essential oil was stored in a dark container with a lid in the
refrigerator, which was covered completely with aluminum foil to avoid any light exposure.
The essential oil accounted for nearly three percent of the seed weight. In order to identify
the active component present in the essential oil, the trans-anethole content was measured
using gas chromatography–mass spectrometry (Agilent GC-Mass 6890N, Agilent Co.,
Santa Clara, CA, USA). The analysis displayed that around 33.40% of the essential oil was
composed of trans-anethole (Table 1). This study was designed as a completely randomized
design in three experimental treatments of 200, 400, and 600 mg/kg of fennel extract in diet
and a control treatment (without essence) in three repetitions.
Table 1. Volatile compounds present in essential oil of Foeniculum vulgare.
Compounds Essential Oil (%) KI
α-Pinene 1.96 914
β-Pinen 0.95 969
Limonen 7.31 1036
1,8 Cineol 7.54 1042
Trepinen 0.81 1093
Fenchone 7.05 1101
Camphor 0.65 1152
4-Terpineol 0.33 1168
α-Terpineol 7.78 1168
Estragloe 13.25 1242
E-Anethole 33.4 1276
2,4-Decadienal 18.12 1326
Germacrene 0.85 1493
2.2. Rearing Condition and Fish Feed
During the experiment, the physicochemical parameters of rearing water, including
temperature, pH, dissolved oxygen, and total hardness, were measured (27
±
1.5
◦
C;
7.3
±
0.21; 7.12
±
0.33 mg/L; and 220.68
±
42.9 mg/L). The light conditions were set as
natural light. Feedstuffs composition and proximate chemical composition of the basal diet
are presented in Table 2.
2.3. Design and Procedure
One hundred twenty pre-productive healthy females of the same weight and same
size (78.66
±
11.10 g and 22.70
±
3.80 cm) were purchased from a local farm. After passing
14 days of adaptation, they were randomly divided into 12 aquariums. The fish were fed
a basic diet during the adaptation period. Food was prepared as follows: the extract was
mixed with the basal diet according to the desired amount, dried at room temperature,
packed, and stored in the refrigerator. Feeding was completed at the rate of 2–3% of body
weight daily for 60 days.
2.4. Sampling
To evaluate the essence’s impact on the growth performance, the fish were fasted
for 24 h at the end of the feeding trial. After anesthetizing the fish with clove powder
(150 mg L
−1
), their weight was measured with a digital scale (ACZET, mp300, Piscataway,
NJ, USA) with an accuracy of 0.01 g [
18
,
19
]. Three fish were chosen randomly from
each repetition and euthanized employing 2 g L
−1
of clove powder. The ovaries were

Water 2023,15, 2978 4 of 13
extracted, weighed, and blood samples were taken using a 2 cc heparin syringe. Growth and
reproduction indices were subsequently computed utilizing the following Formulas [18]:
S peci f ic growt h r ate (SGR)(%body weight day −1)=(lnW f −lnWi)
t×100
Feed conversion ratio (FCR)=Feed consumed
Weight gain
Weight gain (g)=(f ina l weig ht −initial weight)
Gonadosomatic index (GSI)(%)=gonad weight
f ish t otal weig ht −gonad weight ×100
Table 2. Feedstuffs composition and chemical characteristics of the basal diet.
Feed Items Percentage (%)
Basic Diet Experimental
Diet 1
Experimental
Diet 2
Experimental
Diet 3
Fish meal 33 33 33 33
Meat meal 9 9 9 9
Wheat gluten 10 10 10 10
Hydrolyzed yeast 4 4 4 4
Wheat flour 34 34 34 34
Rice bran 3.5 3.5 3.5 3.5
Fish oil 2 2 2 2
Mineral supplement 1.5 1.5 1.5 1.5
Vitamin supplement 1.5 1.5 1.5 1.5
Bentonite 0.5 0.48 0.46 0.44
Sodium chloride 0.5 0.5 0.5 0.5
Antifungal 0.5 0.5 0.5 0.5
Fennel extract 0 0.02 0.04 0.06
Chemical Composition (% of Dry Matter)
Dry matter 94.5 94.68 94.78 94.87
Crude protein 32.5 32.56 32.63 32.59
Crude fat 6.5 6.51 6.52 6.52
Ash 4.5 4.49 4.5 4.49
Crude fiber 7.5 7.48 7.53 7.54
Nitrogen free extract 49 49 49 49
Gross energy (kcal/kg) 3656 3663 3670 3667
2.5. Plasma Biochemical Indices
The prepared blood samples were centrifuged at 6000
×
gfor 5 min using a refrigerated
centrifuge. The plasma was then separated using a sampler and kept in a freezer at
−
80
◦
C
until the test.
The activity levels of aspartate aminotransferase (AST), alanine aminotransferase
(ALT), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) enzymes were
measured using the kits provided by the Pars Azmoun Company and Unico UV/VIS
spectrophotometer (Suite E Dayton, NJ, USA) 2100 [19].
The glucose was measured using the enzyme method of glucose oxidase, while choles-
terol and triglycerides were measured using CHO-PAP [
20
,
21
]. Total plasma protein was
measured based on a biuret reaction at a wavelength of 540 nm [
19
,
21
]. The Bromocresol
Green method was employed to measure plasma albumin levels at 540 nm. Globulin level
was also calculated based on the difference between total protein and albumin levels. The
JAFFE method was used to measure creatinine levels at 510 nm, utilizing picric acid in an
alkaline environment [19,21].

Water 2023,15, 2978 5 of 13
2.6. Oxidative Biomarkers
Catalase (CAT) activity was evaluated based on Góth’s procedure [
22
], which utilized
hydrogen peroxide as a substrate and ammonium molybdate to halt the reaction. On
the other hand, superoxide dismutase, glutathione peroxidase, and glutathione reduc-
tase activities were measured with Biorex biochemical kits following the manufacturer’s
instructions [23].
2.7. Measurement of Sexual Hormones
The concentration of sex steroid hormones (testosterone and estradiol) was measured
through a radioimmunoassay following the guidelines. In order to do so, 50 to 100 micro-
liters of plasma samples, controls, or standard solutions were mixed with rat polyclonal
antibodies coated tubes. Hormones labelled with iodine 125, such as estradiol (COAT-A-
COUNT, Diagnostic Products Corporation, Los Angeles, CA, USA, DPC
®
) or 17 alpha
hydroxyprogesterone (COAT-A-COUNT, Diagnostic Products Corporation, Los Angeles,
CA, USA, DPC®) were added to all test tubes. The mixture was then incubated in a water
bath and washed with phosphate buffer before reading the level of radioactive activity
using a gamma counter.
2.8. Statistical Analysis
The percentage data were converted to Arcsin before conducting a statistical analysis
through SPSS version 18. After ensuring that the data met the two primary requirements
of parametric tests, namely homogeneity of variance and normality, a one-way analysis
of variance test was conducted to explore the main factors and their mutual influence.
Furthermore, Duncan’s multiple range test was conducted at a significance level of 5% to
check for differences in means. Charts and tables were prepared using Excel version 2013.
3. Results
The results of the growth performance of C. carpio following a 60-day feeding study
are shown in Table 3. There was no significant difference in initial weight. The experimental
groups had a significantly greater final weight and weight gain (p< 0.05) compared
to the control group. The highest reported weight gain and final weight were in the
200 mg/kg group.
Table 3.
Growth parameters of C. carpio fed experimental diets containing varying quantities of
F. vulgare essence over 60 days (mean ±SD, n= 3).
F. vulgare Essence Levels (mg/kg Feed)
0 200 400 600
Initial weight 79.25 ±12.34 77.20 ±12.08 78.25 ±12.80 79.93 ±14.32
Final weight 89.20 ±16.32 a93.81 ±18.20 c91.10 ±15.71 b90.85 ±10.4 b
Weight gain 09.87 ±0.21 a16.40 ±0.54 c12.84 ±1.96 b10.92 ±1.24 b
FCR 1.86 ±0.11 bc 1.43 ±0.25 a1.55 ±1.14 ab 2.00 ±0.07 c
SGR 0.25 ±0.17 a0.48 ±0.05 b0.41 ±0.10 ab 0.29 ±0.05 ab
Notes: Significant differences exist between means with different letters in the same row (ANOVA, p< 0.05). FCR:
feed conversion ratio; SGR: specific growth rate.
In addition, the results indicate that oral treatment of 200 mg/kg F. vulgare essence
considerably lowered the FCR. The 600 mg/kg group exhibited the highest FCR value,
with a significant increase compared to the other two experimental groups (p< 0.05). SGR
was significantly higher than the control group (p< 0.05). Table 4displays the mean liver
enzyme activity of the C. carpio that were fed experimental meals, including F. vulgare
essence for 60 days. AST in the control group did not differ substantially from the lowest
treatment (200 mg/kg). However, this value increased significantly (p< 0.05) in the 400 and
600 mg/kg treatments compared to the control group. The lowest ALT activity was then
detected at 200 mg/kg, although the difference was not significant compared to the control.

Water 2023,15, 2978 6 of 13
The maximum ALT activity was observed after the final treatment (600 mg/kg). Regarding
ALP, no significant change was noticed between the control and other treatments. LDH
activity increased significantly at 600 mg/kg compared to the control, but there was no
significant difference between the control and the other treatments. Table 4represents the
mean plasma biochemical parameters of the C. carpio that were fed the experimental diets
for 60 days. The results showed that glucose and triglyceride did not change significantly
compared to the control and the treated groups. Total protein was significantly increased
in the 400 and 600 mg/kg treatments compared to the control (p< 0.05). The highest and
lowest albumin contents were reported in the 600 and 200 mg/kg treatments, respectively
(p< 0.05). No significant difference was observed between the albumin content of the
control group and the 400 mg/kg treatment. A significant increase in globulin value
was detected at 400 mg/kg compared to the control (p< 0.05). Other treatments had no
significant difference from the control. Cholesterol content was not significantly changed
and remained unaffected. The plasma creatinine content, only at the highest level of
600 mg/kg, showed a significant increase compared to the control (p< 0.05).
Table 4.
Plasma biochemical parameters of C. carpio that were fed the experimental diets containing
different levels (0, 200, 400, and 600 mg/kg) of F. vulgare essence for 60 days (mean ±SD, n= 3).
F. vulgare Essence Levels (mg/kg Feed)
0 200 400 600
AST (U/L) 48.57 ±6.14 a53.22 ±4.65 ab 55.71 ±2.71 b58.86 ±3.75 b
ALT (U/L) 15.92 ±1.15 ab 13.16 ±0.55 a14.92 ±0.98 b16.39 ±0.94 c
LDH (U/L) 416.68 ±19.64 a441.14 ±39.76 ab 462.78 ±78.47 ab 484.22 ±27.27 b
ALP (U/L) 184.71 ±8.35 181.35 ±10.71 193.42 ±14.48 190.48 ±13.89
Glucose (mg/dL) 64.94 ±4.33 65.03 ±10.72 60.99 ±5.59 67.11 ±10.21
Total protein (g/dL) 2.71 ±0.14 a2.23 ±0.50 a3.45 ±0.60 b3.53 ±0.64 b
Albumin 2.00 ±0.15 b1.24 ±0.19 a1.86 ±0.08 b2.48 ±0.46 c
Globulin (g/dL) 0.71 ±0.11 a1.00 ±0.39 a1.59 ±0.60 b1.05 ±0.53 a
Cholesterol (mg/dL) 73.80 ±4.87 ab 80.18 ±5.55 b67.57 ±8.41 a69.80 ±6.05 a
Triglycerides (mg/dL) 185.9 ±17.25 173.75 ±12.06 168.34 ±15.52 171.33 ±14.43
Creatinine (mg/dL) 0.31 ±0.04 a0.36 ±0.11 a0.40 ±0.11 a0.53 ±0.10 b
Notes: Significant differences exist between means with different letters in the same row (ANOVA, p< 0.05). AST:
aspartate aminotransferase; ALT: alanine aminotransferase; LDH: lactate dehydrogenase.
The mean plasma biochemical parameters of C. carpio that were fed the experimental
diets for sixty days are shown in Table 4. Glucose and triglyceride levels did not differ
significantly between the control and experimental groups. The 400 mg/kg and 600 mg/kg
treatments significantly enhanced total protein compared to the control (p< 0.05). The
treatments with the highest and lowest albumin levels were 600 mg/kg and 200 mg/kg,
respectively (p< 0.05). The difference between the control group and the 400 mg/kg treat-
ment was not statistically significant. The group with 400 mg/kg exhibited a substantial
rise in globulin levels compared to the control (p< 0.05). Other treatments did not differ
significantly from the control. The cholesterol content did not change considerably and
stayed unchanged. Only at the highest dosage, 600 mg/kg, did plasma creatinine levels
increase significantly compared to the control (p< 0.05).
As shown in Table 5, plasma CAT activity remained unaltered with experimental diets;
however, SOD activity decreased significantly in the 600 mg/kg group compared to the
control group (p< 0.05). Compared to the control, the plasma GPx activity of the treated
groups significantly decreased (p< 0.05).

Water 2023,15, 2978 7 of 13
Table 5.
Antioxidant enzyme activity of C. carpio that were fed the experimental diets containing
different levels (0, 200, 400, and 600 mg/kg) of F. vulgare essence for 60 days (mean ±SD, n= 3).
F. vulgare Essence Levels (mg/kg Feed)
0 200 400 600
CAT (KU/mg protein) 0.15 ±0.01 0.15 ±0.02 0.14 ±0.03 0.14 ±0.03
SOD (U/mg protein) 0.78 ±0.25 b0.90 ±0.24 b0.70 ±0.32 b0.36 ±0.07 a
GPx (U/mg protein) 7.80 ±1.06 c4.45 ±1.45 b2.51 ±0.75 a2.66 ±0.46 a
Note(s): Significant differences exist between means with different letters in the same row (ANOVA, p< 0.05).
CAT: catalase; SOD: superoxide dismutase; GPx: glutathione peroxidase.
Estradiol levels in female C. carpio that were fed 400 mg/kg were substantially greater
than the control (Figure 1a, p< 0.05), although the other treatments were unchanged. The
investigation of testosterone concentration, on the other hand, revealed that the highest
concentration was found in carps that were fed 200 mg/kg F. vulgare essence, which was
significantly greater than the control (Figure 1b, p< 0.05). Compared to the control, the
higher inclusion levels had no significant impacts.
Water 2023, 15, x FOR PEER REVIEW 7 of 13
stayed unchanged. Only at the highest dosage, 600 mg/kg, did plasma creatinine levels
increase signicantly compared to the control (p < 0.05).
As shown in Table 5, plasma CAT activity remained unaltered with experimental di-
ets; however, SOD activity decreased signicantly in the 600 mg/kg group compared to
the control group (p < 0.05). Compared to the control, the plasma GPx activity of the
treated groups signicantly decreased (p < 0.05).
Estradiol levels in female C. carpio that were fed 400 mg/kg were substantially greater
than the control (Figure 1a, p <0.05), although the other treatments were unchanged. The
investigation of testosterone concentration, on the other hand, revealed that the highest
concentration was found in carps that were fed 200 mg/kg F. vulgare essence, which was
signicantly greater than the control (Figure 1b, p < 0.05). Compared to the control, the
higher inclusion levels had no signicant impacts.
Table 5. Antioxidant enzyme activity of C. carpio that were fed the experimental diets containing
dierent levels (0, 200, 400, and 600 mg/kg) of F. vulgare essence for 60 days (mean ± SD, n = 3).
F. vulgare Essence Levels (mg/kg Feed)
0
200
400
600
CAT (KU/mg protein)
0.15 ± 0.01
0.15 ± 0.02
0.14 ± 0.03
0.14 ± 0.03
SOD (U/mg protein)
0.78 ± 0.25 b
0.90 ± 0.24 b
0.70 ± 0.32 b
0.36 ± 0.07 a
GPx (U/mg protein)
7.80 ± 1.06 c
4.45 ± 1.45 b
2.51 ± 0.75 a
2.66 ± 0.46 a
Note(s): Signicant dierences exist between means with dierent leers in the same row (ANOVA,
p < 0.05). CAT: catalase; SOD: superoxide dismutase; GPx: glutathione peroxidase.
Figure 1.
Estradiol (
a
) and testosterone (
b
) concentration in plasma and a gonadosomatic index (
c
) in
C. carpio that were fed the experimental diets containing different levels (0, 200, 400, and 600 mg/kg)
of F. vulgare essence for 60 days (mean
±
SD, n= 3). Different letters indicate significant differences
(p< 0.05).

Water 2023,15, 2978 8 of 13
Figure 1c displays the GSI of C. carpio that were fed varying concentrations of F. vulgare
essence. After 60 days of therapy, the GSI of the experimental groups (200 and 400 mg/kg)
increased significantly compared to the control group (p< 0.05). No significant differences
were identified between the tested groups. The highest treatment (600 mg/kg) represented
no significant increase compared to the control.
4. Discussion
Phytoestrogens are plant-derived compounds that can mimic the effects of estrogen
in animals. Some studies have explored the use of phytoestrogens in inducing fish to
ovulate, particularly in species where spawning can be difficult to induce. However, it is
important to note that the use of phytoestrogens in aquaculture is still a relatively new
area of research, and there are concerns about the potential environmental impacts of
increased phytoestrogen levels in aquatic ecosystems. Additionally, the effectiveness of
phytoestrogens in inducing ovulation may vary depending on fish species and other factors,
so further studies are needed to fully understand their potential applications in aquaculture.
Phytoestrogens may be excreted, absorbed, or broken into stronger compounds after
entering the digestive tract. Isoflavones become active during digestion as well as absorp-
tion in human and animal bodies, where this conversion is processed by bacteria in the
small intestine as follows: the sugar part is separated from the molecules by bacteria and
converted into an active form. Then, the activated form of isoflavones is absorbed from
the small intestine, most of which is taken up by the liver after entering the body. A small
amount is excreted by the kidneys and through urine [
24
]. Absorbed phytoestrogens and
isoflavones can interfere with the expression of growth-related genes such as IGF1 and
promote growth [
25
]. Since fennel essence has antimicrobial properties [
26
], the presence
of this compound in the intestines of the treated fish will change the microbial balance of
the intestines in favour of the host and will help the growth of the host by enhancing the
production of digestive enzymes. Such results were also observed in the use of other herbal
compounds such as peppermint extract [27] and Iranian shallot extract [28].
Based on the results, fennel essential oils improved growth, FCR, and SGR in common
carp (C. carpio). Many studies revealed the positive effects on fish growth with the oral
administration of plant-based essential oils. For instance, according to Kesbic et al. [
29
], the
administration of a Monterey Cypress (Cupressus macrocarpa hartw) leaf essential oil as a
dietary supplement of C. carpio’s diet was found to be a suitable growth promotor. Moreover,
similar results to our findings were obtained in the treatment of Oreochromis niloticus and
Poecilia reticulate with fennel [
17
,
30
]. Studies have shown that a Bergamot (Citrus bergamia)
peel oil supplementation in fish diets was able to optimize growth performance, feed
utilization, and general health status [
31
,
32
]. Vitex agnus extract, which has phytoestrogen
properties, was used to produce feminization, with the results indicating that 15 g/kg of
the extract also increased growth [33].
Phytoestrogens can react with enzymes and receptors, and due to their stable struc-
ture and low molecular weight, they can pass through cell membranes [
34
,
35
]. There is
evidence confirming that phytoestrogens affect fat metabolism. Since fats play an essential
role in supplying the nutrients in the eggs, phytoestrogens can likely help improve the
storage of nutrients in the developing ovaries and ultimately enhance the GSI as well as
accelerate the sexual maturation of fish. The results of our study also showed that fennel
essence significantly contributed to the development of gonads in the treatments of 400 and
200 mg/kg. There are various studies indicating the positive effect of phytoestrogens
in boosting vitellogenin production and improving GSI. For example, researchers who
investigated the effect of fennel essential oils and phytoestrogen genistein in Cichlasoma
nigrofasciatum and Huso huso, respectively, achieved similar results at the level of 125 mg/kg
and 0.4 g/kg, respectively [6,16].
Phytoestrogens bind to estrogen receptors in the body with less affinity than estradiol;
they are weakly bound to ER
α
receptors and strongly bound to ER
β
receptors with specific
and anti-estrogenic effects, in such a way that they have agonist effects in some tissues

Water 2023,15, 2978 9 of 13
and have antagonistic effects in others. On the other hand, phytoestrogens that bind to
ER
β
induce the transcription of estrogen target genes more than when they are bound to
ER
α
[
33
] and can leave estrogen agonistic plus antagonistic effects. In this study, it was
also found that fennel seed essence did not influence increasing testosterone production,
but it significantly elevated estradiol in the 400 mg/kg treatment. There are several studies
indicating the effect of plant compounds containing phytoestrogens, such as vitex [
31
],
Tribulus terrestris [
34
,
35
], garlic [
36
], aloe vera [
37
], and Matricaria recutita [
7
], on the sexual
performance of fishes. They have revealed that these plants can exert effective hormone-like
effects on the fish in certain amounts due to the presence of estrogen-like compounds. The
effect of fennel in enhancing the secretion of sex hormones in P. reticulata and rats [
17
,
38
]
has also been proven.
In general, it can be stated that the activity of phytoestrogens in the body depends
on factors such as the concentration of estrogens in the body, the state of saturation of
estrogen receptors, the duration of binding of phytoestrogens to estrogen receptors, and
the time it takes until the phytoestrogen is broken down and enters the blood circulation.
When the concentration of estrogen in the blood is low, phytoestrogens show pro-estrogen
effects, while on the contrary, when the concentration of estrogen in the blood is high,
they have anti-estrogenic effects and impact the growth of estrogen-dependent cells plus
the sexual cycle [
38
]. The results of this study also indicated that fennel seed essence at
the highest level (600 mg/kg ration) both reduced the amount of estradiol and prevented
gonad growth. Similar results were obtained in the study of the effect of this plant on the
development of the testes of common carp, where the excessive dietary administration of
the extract caused a decline in GSI and plasma testosterone concentration [39].
An increase in the activity of liver enzymes is a sign of unfavourable rearing conditions
and vital organ tissue damage such as the liver and kidney, so measuring the activity of liver
enzymes has always been mentioned as an indicator of fish health, and their investigation
is recommended in research related to medicinal plants. Fennel seed essence has been
reported to have hepatoprotective activity [
8
]. The results of a biochemical analysis of
plasma compounds showed that the treatment of carp with fennel seed essence had no
significant effect on liver enzymes, and only ALT activity was significantly reduced in the
treatment of 200 mg/kg. These results suggest that fennel essential oil in the amounts that
were used did not damage the tissue but protected it.
Reactive oxygen species (ROS) are key signalling molecules that play an important
role in healing inflammatory disorders and help clear pathogens as well as foreign particles.
However, high levels of reactive oxygen species can also destroy normal cells [
40
], while
antioxidant enzymes can defend against this excessive increase in these molecules [
13
]. One
of the reasons for using medicinal plants in the ratio of aquatic animals is to boost the an-
tioxidant capacity of the body and improve the ability to cope with adverse environmental
conditions, which ultimately enhances survival and production in aquaculture. SOD, CAT,
Gr, and GPx are the main antioxidant enzymes that catalyze reactive oxygen species [
41
,
42
].
Despite the strong antioxidant activity in fennel seed extract [
43
,
44
], there have not been
many studies on the effect of this plant on the antioxidant activity of aquatic animals. In
the present experiment, the results confirmed the increase in SOD enzyme activity, while
glutathione peroxidase decreased significantly in the experimental treatments. Similar to
our findings, the activity of antioxidant enzymes and the expression level of SOD plus
CAT genes increased significantly in the liver of Micropterus salmoides treated with fennel
essence [13].
In this experiment, the amount of total protein, albumin, globulin, and cholesterol
increased due to treatment with fennel seed essence, and no change was reported in glucose
and triglyceride status. The biochemical parameters of blood reflect the health, nutritional,
and environmental status of the fish, where the change in the biochemical variables of
blood is probably the result of increasing the non-specific immune response of fish [
45
],
since globulin, albumin, and total protein are known as important components of the innate
immune system of fish [
23
]. Consistent with our findings, Gulec et al. [
46
] reported that

Water 2023,15, 2978 10 of 13
the administration of fennel seed essential oils to the Oncorhynchus mykiss diets elevated
plasma biochemical indices, including total protein, albumin, cholesterol, triglycerides,
and bilirubin.
There are various studies that confirmed the reduction in glucose due to the use of
fennel [
42
,
44
] and other plants containing phytoestrogens such as Vitex [
29
], while the
glucose level remained constant in our experiment. Glucose reduction can be due to
increased insulin secretion or increased fish metabolism because of drug treatments [
24
],
whereas fennel apparently did not show such an effect. Cholesterol, as a precursor of
steroid hormones, plays an important role in the biosynthesis of these hormones and the
acceleration of sexual maturation. The increase in the level of blood cholesterol as well as
the increase in the secretion of estradiol hormone both confirm the positive effects of fennel
on the sexual activity of carps.
Creatinine is a breakdown product of creatine phosphate resulting from protein
metabolism, which is released by the body at a constant rate [
47
]. Blood creatinine is
an important indicator of kidney health, as it is an easily measurable byproduct of muscle
metabolism excreted by the kidneys without any change [
47
]. In the present study, the
amount of blood creatinine in the treatments of 200 and 400 mg/kg was not significantly
different from the control group, indicating that this level of essence did not negatively
affect the kidney and vital organs, but the 600 mg/kg treatment was associated with a
notable increase in the creatinine content. Studies by Abdel Rahman et al. [
48
] showed
that in the case of aflatoxin poisoning in Oreochromis niloticus, fennel essential oils can
significantly lower the level of blood creatinine, which was elevated due to poisoning.
It is crucial to mention that phytoestrogens can negatively impact species and aquatic
ecosystems that are not the intended target. Studies showed that phytoestrogens could
have multiple effects on aquatic ecosystems. Phytoestrogens could disrupt the endocrine
system in aquatic animals, alter their behaviours, and change population dynamics. More-
over, waterborne phytoestrogens could impact non-target species and cause shifts in their
community structure [
49
,
50
]. Therefore, it is recommended to investigate its unwanted
environmental effects before prescribing any phytoestrogen.
5. Conclusions
The findings from our research demonstrated that the inclusion of fennel essential oil
in the dietary regimen of C. carpio not only enhanced their reproductive capabilities but also
had positive effects on their overall health and antioxidant status. A noteworthy aspect of
this study was the careful consideration given to the amount of medicinal plant compounds
utilized in the aquatic diet. The results indicated that excessive use of these products could
exert undue pressure on vital organs such as the liver and kidneys, potentially leading to
adverse effects on reproduction. Hence, it is important to strike a balance. Considering all
factors, a recommended dosage of 200 mg/kg of fennel seed essential oil is suggested for
optimal carp breeding outcomes.
Author Contributions:
Conceptualization, H.A.M., Y.H. and M.B.; methodology, H.A.M.; software,
H.A.M. and M.B.; validation, H.A.M., Y.H. and M.B.; formal analysis, H.A.M. and H.R.; investigation,
H.A.M. and Y.H.; resources, H.A.M., Y.H. and M.B.; data curation, H.A.M. and Y.H.; writing—original
draft preparation, H.A.M. and M.B.; writing—review and editing, C.R.M. and C.F.; visualization,
H.A.M., Y.H., M.B. and C.R.M.; supervision, M.B. and C.F.; project administration, M.B; funding
acquisition, H.A.M. All authors have read and agreed to the published version of the manuscript.
Funding:
This work was supported by Hamidreza Ahmadniaye Motlagh’s personal research grant
support from the Ferdowsi University of Mashhad.
Institutional Review Board Statement:
All the experiments were based on the instructions for
working with laboratory animals at the Ferdowsi University of Mashhad.
Data Availability Statement: All data that created were presented as Tables and Figures.
Conflicts of Interest: The authors declare no conflict of interest.

Water 2023,15, 2978 11 of 13
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