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Received: 16 December 2021 Revised: 5 May 2022 Accepted: 10 May 2022

DOI: 10.1002/aff2.59

ORIGINAL ARTICLE

The efficacy of using pine (Pinus massoniana) pollen as an

alternative to synthetic steroids in producing monosex male
Nile tilapia (Oreochromis niloticus, L.)

Md. Abdul Aziz1 Mst. Momtaz Mostary1 Israt Jahan Sume1 Md. Helal Uddin2
Mohd Golam Quader Khan1 Md. Samsul Alam1 M. Sadiqul Islam3

1
Department of Fisheries Biology and
Genetics, Bangladesh Agricultural University, Abstract
Mymensingh, Bangladesh
Synthetic sex steroids are widely used to produce all-male stocks in Nile tilapia
2
Department of Fisheries Management,
(Oreochromis niloticus). However, there is a growing concern about the use of synthetic
Bangladesh Agricultural University,
Mymensingh, Bangladesh steroid hormones in fish as food. We investigated the efficacy of a natural sex hormone
3
Department of Marine Fisheries Science, as an alternative to synthetic steroids in producing monosex male tilapia. Two-day-
Bangladesh Agricultural University,
Mymensingh, Bangladesh
old fry were fed pine pollen powder at three different concentrations (0.5%, 1.0%,
1.5%) for 28 days and subsequently reared for 3 months to observe the efficacy of
Correspondence
pine pollen as well as to optimize the concentration for sex reversal. As a positive con-
M. Sadiqul Islam, Department of Marine
Fisheries Science, Bangladesh Agricultural trol, same-aged fry of three different commercial tilapia hatcheries were fed synthetic
University, Mymensingh-2202, Bangladesh.
steroid and 17-α-methyl testosterone-treated feed (60 mg/kg) for the same duration.
Email: sadiqul.mfs@bau.edu.bd
Before analyzing the phenotypic sex, the fish were reared on untreated commercial
Md. Abdul Aziz and Mst. Momtaz Mostary feed for 90 days in separate tanks. The 1.0% pine pollen treatment yielded a mean
contributed equally to this study, and both
should be considered first authors. of 92.50 ± 0.51% males among the progeny, higher than the mean (89.49 ± 0.25%)
obtained from synthetic steroid treatments. This result demonstrates that pine pollen
Funding information
could be an effective compound for the direct masculinization of Nile tilapia. We also
Ministry of Science & Technology
obtained a significant difference (p < 0.05) in the growth performance and survival of
tilapia using the dietary allotment of pine pollen. Our study suggests the feasibility of
this compound as an eco-friendly method of masculinization of tilapia and can be tried
in commercial aquaculture practice.

KEYWORDS
androgenic herb, Nile tilapia, pine pollen, sex reversal, synthetic hormone, testosterone

1 INTRODUCTION revolutionized fish farming worldwide (Ghosal & Chakraborty, 2017)

since females of tilapia are prolific breeders with attainment of sex-
Hormonal sex reversal is a technique of changing sex by administer- ual maturity at an earlier age (4–6 months) with slower growth. Mixed
ing hormones before or during the period of sexual differentiation. sex culture exhibits stunted somatic growth of the progeny through
Tilapia sex reversal, the technology for producing all male tilapia, has unwanted reproduction throughout the year. In contrast, the male-only

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2022 The Authors. Aquaculture, Fish and Fisheries published by John Wiley & Sons Ltd.

Aqua. Fish & Fisheries. 2022;375–383. wileyonlinelibrary.com/journal/aff2 375


376
tilapia culture is preferred by farmers for increased productivity due to
faster growth of males as well as to control unwanted reproduction by
females (Bardhan et al., 2021).
A highly effective method for inducing sex reversal in tilapia and
tilapia hybrids is the use of synthetic androgen hormones in the early
life stages of tilapia fry (Guerrero & Guerrero, 1988). Methods such as
introducing androgens to fish by oral administration and fry immersion
in steroid solutions (Fuentes-Silva et al., 2013) have been used. In addi-
tion, extracts from some plants, for example, Basella alba and Tribulus
terrestris, have been used in producing a predominantly male popula-
tion of tilapia in aquaculture (Gabriel et al., 2017; Ghosal et al., 2015;
Khakong et al., 2011).
Anabolic steroids are best used in specific situations and within
a limited time, for example, to help rebuild severely injured tissues
and treat certain types of diseases (e.g., angioedema). However, there
are fears and debates regarding the use of synthetic steroids on
the grounds of potential health risks, for example, on vital organs
(liver, kidney, pancreas, and gills), metabolic profiles, and nucleic
acids (Davani-Davari et al., 2019). In the environment, steroids are
either biodegraded or mineralized. However, the escape of fish from
hatcheries into natural water may alter the dynamics of the envi-
ronment due to unforeseen consequences (Megbowon & Mojekwu,
2014).
In tilapia, the anabolic steroid methyltestosterone (MT) is usu-
ally used for inductive sex reversal, but the disadvantage is that it
is expensive and a growth inhibitor (Jensi et al., 2016; Suseno et al.,
2020). Alternative methods have been investigated to produce mono-
sex tilapia in recent years, for example, the production of all genetically
male tilapia sired by putative YY males (Sultana et al., 2020). However,
the success of such a method has been hindered by the very complex
sex determination system of tilapia (Baroiller et al., 2009; Kobayashi
et al., 2013). Another option is to use dietary organic androgens, which
are more easily accepted by consumers and are mainly derived from
natural sources.
Phytochemicals such as pigments, alkaloids, terpenoids, flavonoids,
phenolics, steroids, and essential oils are a large group of plant-derived
compounds that have been reported to assist several activities, such
as appetite stimulation, growth promotion, endocrine modulation,
immunostimulation, and stress relief (Chakraborty et al., 2011, 2013).
Despite the notable benefits to health and performance that phyto-
chemicals bring in for various terrestrial animals, their use in fish farm-
ing has rarely been investigated. Some phytochemicals are reported to
block biosynthesis and the action of oestrogen by acting as aromatase
inhibitors and antagonists to nuclear oestrogen receptors in gonad
germ cells (Gabriel & Redondo, 2019) and hence could be considered
potential compounds in inducing sex reversal in fish.
Raw pine pollen (Pinus massoniana) is a Chinese medicinal herb with
an androgenic effect that has been used as a drug for colds, prostate
disease, anaemia, diabetes, hypertension, asthma, and rhinitis, as well
as for food for thousands of years (Adenigba et al., 2017; Lee et al.,
2009).
A different species of pine, Pinus tabulaeformis, has also been
used as a replacement for 17-alpha-methyltestosterone in the diet
of Nile tilapia larvae for sex reversal and growth (Nian et al., 2017).
26938847, 2022, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/aff2.59 by Turkey Cochrane Evidence Aid, Wiley Online Library on [10/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
AZIZ ET AL .
Pine pollen contains all four major androgen (male sex) hormones,
androstenedione, testosterone, dehydroepiandrosterone (DHEA), and
androsterone (Danuše, 2019). Because androgen hormones are
present in pine pollen, once digested or absorbed, pine pollen raises
testosterone levels almost immediately (Wong, 2019). The sterols
present in pine pollen can raise the testosterone levels in the blood
and balance the ratio of androgens to oestrogen. The addition of such
organic dietary hormones may provide a great boost to profitability
in aquaculture by effectively increasing growth rates, feed intake, or
food conversion efficiencies. The present study aimed to determine
the optimum dose of pine pollen as well as to observe the efficacy of
pine pollen as a worthy substitute for 17-α-MT to induce sex reversal
(masculinization) with its anabolic effects in Nile tilapia, Oreochromis
niloticus.
2 MATERIALS AND METHODS
2.1 Experimental site, approach, and ethical
statement
The primary experimental site of the present study was the Wet
Laboratory of the Department of Fisheries Biology and Genetics,
Bangladesh Agricultural University, Mymensingh. In parallel with the
experimental setup in the primary site, three private fish hatcheries
were chosen where synthetic androgen hormone (17-α-methyl testos-
terone, MT) was routinely used for commercial monosex (male) pro-
duction of Nile tilapia. These were Farm 1 (Agro-3 Fish Hatchery and
Culture Farm, Trishal), Farm 2 (Fishtech Hatchery Ltd, Tarakanda), and
Farm 3 (Sharnalata Agro Fisheries Ltd, Fulbaria) of Mymensingh dis-
trict in Bangladesh. The study was performed in two phases. First, the
effects of different doses of pine pollen in terms of sex reversal and
growth parameters were observed at the primary site to optimize the
dose. Second, the efficacy of the optimized pine pollen treatment was
compared with the efficacy of the synthetic steroid 17-α-MT routinely
used in the above three farms. To facilitate the collection and com-
pilation of data, some experiments were run in parallel. The animal
scientific procedure was rigorously followed with prior approval by the
animal welfare and the ethics committee of Bangladesh Agricultural
University.
2.2 Optimization of pine pollen for growth and
masculinization of tilapia
2.2.1 Collection and acclimation of fish fry and
experimental setup
Newly hatched (2days old) tilapia fry were collected from the freshwa-
ter station of Bangladesh Fisheries Research Institute in an oxygenated
bag and transported to the wet laboratory of the Faculty of Fisheries,
Bangladesh Agricultural University, where they were acclimated for
24 h in a fine meshed hapa set in a cistern. Eight glass aquariums
(each 45 cm × 30 cm × 30 cm) of 30 L capacity each were used for

AZIZ ET AL .
stocking 130 fry/aquarium for pine pollen treatments. Underground
water was stored in cisterns to be used during fry rearing and for
periodical replenishment. Water quality parameters were routinely
checked and recorded from prior to the stocking of fry in hapa for
acclimation to the end of the experimental period (pH by portable pH
meter HANNA, HI98107, RO; DO by DO meter, YSI, DO200A, US;
others by routinely used equipment and test kit) to ensure suitable
range of the parameters, that is, DO 3.9–8.5 ppm, pH 6.8–8.5, chlorine
0 ppm, nitrate 10–40 ppm, nitrite 0–0.5 ppm, hardness 80–120 ppm,
and alkalinity 40–100 ppm.
2.3 Experimental diet preparation, pine pollen
treatment, and basic maintenance
An androgenic herb, pine pollen (P. massoniana) (Raw Organic Bro-
ken Cell Wall Pine Pollen Powder, CN), was procured and homo-
geneously mixed (using 70% ethanol for a mixer of 1 kg feed and
subsequently sundried) with commercial feed (Mega feed Co., BD) at
rates of 0.5% (500 mg/kg feed), 1.0% (1000 mg/kg feed), and 1.5%
(1500 mg/kg feed). The proximate composition of the feed used was
29.05% protein, 4.33% lipid, 13.42% ash, 5.40% crude fibre, 32.42%
carbohydrate, and 15.39% moisture. The control group (C) feed con-
tained no (0%) pine pollen. Feeding regimes were strictly followed as
given: five times (8.00, 11.00, 14.00, 17.00, 20.00) a day for 10 days, ad
libitum, followed by four times (10.00, 13.00, 16.00, 19.00) a day for the
next 20 days and three times a day for the subsequent period of rear-
ing. The water temperature was maintained at 28◦ C ± 0.5◦ C. Everyday
siphoning (with replenishment of siphoned out water) was performed,
24 h of aeration was maintained, and mortalities (if any) were recorded.
2.4 Observation of growth parameters, survival
(%), and sex
Fish were sampled weekly to obtain growth parameters, that is, % gain
in length and weight. The specific growth rate (%/day, with follow-
ing formula) and survival at the end of the hormone treatment were
also determined; SGR = (ln(final weight in grams) – ln(initial weight in
grams) × 100)/t (in days).
Feeding (pine-pollen free) was continued for an additional 90-day
period, three times a day, ad libitum, before being sexed by the aceto-
carmine staining method (Guerrero & Shelton, 1974). Gonads with
mature cells of spermatogonia, oogonia, and intersex cells (spermato-
oocytes) were observed, and photographs were captured (OPTICA
B350, IT) through compound microscopy.
2.5 Observation of growth performances,
survival, and sex ratios in optimized pine pollen and
MT-treated groups
Fry of similar age (2 days old) underwent synthetic androgen treat-
ments (alongside with optimized pine pollen treatment) for the same
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377
duration (28 days) in all three hatcheries using the same feeding
protocols in their existing setups with the in-practice dose of MT
(10 mg/kg feed) for commercial monosex production.
2.6 Post-MT-treated fry rearing
One-month-old (28 days MT treated) synthetic androgenic fry of Nile
tilapia from three different private fish hatcheries were brought and
reared in the aquaria systems of the wet laboratory, Faculty of Fish-
eries, Bangladesh Agricultural University, along with the optimized
pine pollen-treated group. All batches underwent rearing for another
90 days. During this time, feeding was administered devoid of MT
hormone and pine pollen sterol treatments three times ad libitum.
Basic maintenance rigorously followed scientific procedures, including
continuous aeration of aquaria, regular siphoning and replenishment
by ground water, and weekly testing of water quality parameters to
ensure suitable ranges for the fish.
2.7 Measurement of growth parameters and
determination of sex
All the experimental individuals were weighed and sacrificed using a
lethal dose of anaesthetic (clove oil, 25 mg/L) after 90 days of rear-
ing. All fish were dissected after taking the final length and weight. Sex
determination was performed by macroscopic and microscopic exam-
inations of gonad tissues using the standard aceto-carmine squash
technique (Guerrero & Shelton, 1974).
2.8 Statistical analysis
Nested analysis of variance (ANOVA) was performed to test the vari-
ation among the treatment as well as replica (aquaria) effects on
growth parameters and sex reversals using SPSS v25. The relationship
and differences between treatments (mean ± SD) were determined
using Duncan’s multiple range test at the 5% level of significance.
The deviation of the sex ratio from 1:1 was tested using chi-square
statistics.
3 RESULTS
3.1 Growth, survival, and sex reversal in the pine
pollen groups and comparative analysis with
MT-treated groups
The final mean lengths and weights after 28 days of rearing of the
fry are presented in Table 1, which showed no significant difference
(p > 0.05) among the treatments. Survival (at 120 days) also did
not show any significant differences among the treatments (Table 1).
The proportions of males were higher (p < 0.05) in all three pine
pollen groups than in the untreated pine pollen group (Table 1).
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378 AZIZ ET AL .

The highest percentage of males (92.50 ± 0.51%) was observed with

1.0% pine pollen (p < 0.001), and the lowest was observed in the

Intersex (%)

3.71 ± 0.05

2.50 ± 0.05

2.75 ± 0.68

2.73 ± 1.33
untreated group (52.04 ± 0.36%). The votestes obtained (%) with
and without pine pollen did not vary (p > 0.05). The proportions of
males to females deviated significantly from 1:1 in all pine pollen

92.50 ± 0.51**

81.19 ± 0.89**
72.67 ± 2.32* treatments.

52.04 ± 0.36
In the comparative analysis, performances with the optimal concen-
Male (%)

tration (1.0%) of pine pollen were observed with synthetic androgens.

A higher gain in weight (30 ± 2.00 g) and the SGR (9.20 ± 0.20%)
Growth and survival of tilapia fry (during 28 days) and determination of sex (120 days) with different doses of pine pollen (N = 130/replicate/treatment)

were obtained with the optimized concentration of pine pollen com-

90.00 ± 1.18a

92.31 ± 2.31a

90.84 ± 1.21a

90.08 ± 1.77a
Survivals (%)

pared to the synthetic MT, but with marginally significant differences

(mean ± SD)

(p = 0.0413 and 0.0387 for weight and SGR, respectively) (Table 2).

Notes: The data are presented as the mean ± SD. Different asterisks/superscripts in the same column indicate statistically significant differences from control groups.
While observing the androgenic effect, the highest percentage of
male tilapia (92.50 ± 0.51%) with 1% pine pollen was found to be
significantly different from other groups. Nested ANOVA revealed a
45.50 ± 0.62b

43.80 ± 0.56b

b
10.41 ± 0.06a

10.60 ± 0.05a

11.39 ± 0.07a

11.28 ± 0.05a
42.30 ± 0.69

44.25 ± 0.71

significant effect among the replications (aquaria) as well (p < 0.05).

28 days

Intersex tilapia was evident to some extent in all treatment groups,

with MT-2 (Fishtech) having the highest value of 7.27 ± 0.03% and with
1% having the lowest value of 2.5 ± 0.05%. The percentages of males,
33.75 ± 0.50
10.30 ± 0.18
32.15 ± 0.38
10.90 ± 0.16
36.20 ± 0.54
10.55 ± 0.21
34.40 ± 0.43

females, and intersexes in the different groups are shown in Table 2.

9.85 ± 0.18
21 days

3.2 Gonadal histology with different

concentrations of pine pollen
9.45 ± 0.15

21.65 ± 0.63
9.85 ± 0.18
22.10 ± 0.48
9.75 ± 0.12
23.15 ± 0.50
9.85 ± 0.21
24.30 ± 0.51
Length (mm) and weight (mg) (mean ± SD)
14 days

Microscopic observations of the gonads treated with various concen-

trations of pine pollen are shown in Figure 1. Structural aberrations
in the gonadal cells were not evident with different concentrations of
8.85 ± 0.08

17.84 ± 0.36
9.28 ± 0.13
15.28 ± 0.29
9.65 ± 0.18
16.31 ± 0.43
9.15 ± 0.09
18.23 ± 0.41

pine pollen compared to the control. However, some distorted oocytes

(and oogonia) were noticed with 1.5% pine pollen in the intersex
7 days

compared with other treatments (Figure 1). Incomplete rupturing of

connective tissues of the female gonad was also observed with 1.5%
pine pollen treatment (Figure 1).
Weight

Weight

Weight

Weight
Length

Length

Length

Length

3.3 Observation of gonads under the treatments

of optimized pine pollen (1%) and synthetic
androgen, MT
T1 (0.5% pine pollen)

T2 (1.0% pine pollen)

T3 (1.5% pine pollen)

Microscopic observations of different types of gonads treated with

0% pine pollen

optimized pine pollen and synthetic MT were compared, as shown in

Treatments

Figure 2. Both the male and female gonads obtained with the two treat-
ments (1% pine group and MT) looked normal. Intersex samples were
apparent with spermato-oogonial cells in both groups but without any
noticeable morphological differences.
Initial (I) length (mm) and
weight (mg) (mean ± SD)

IL: 7.71 ± 0.05 and IW:

4 DISCUSSION
7.00 ± 0.13

Our study aimed at finding a suitable alternative (with optimized con-

TA B L E 1

**p < 0.01.

centration/s) to synthetic steroids, which are being used widely in

*p < 0.05.

the direct food chain and hence have created a high level of concern
for consumers in recent years. Although it has been shown that the
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AZIZ ET AL . 379

TA B L E 2 Growth performances and proportions of male, female and intersex Nile tilapia in optimized pine pollen and methyl testosterone
treatments after 120 days (N = 130/replicate/treatment)

Variables Wt (g) in 90 days SGR (%/day) Survival (%) % Male % Female % Intersex
1% pine group 30 ± 2.00 a
9.20 ± 0.20 a
92.31 ± 2.31 a
92.50 ± 0.51 a
5.00 ± 0.20 c
2.50 ± 0.05b
MT1 23 ± 1.00b 8.74 ± 0.20c 90.77 ± 0.77ab 89.83 ± 0.10bc 6.78 ± 0.10a 3.39 ± 0.09b
MT2 23 ± 0.50b
8.88 ± 0.12bc 84.62 ± 0.62bc 88.18 ± 0.18c 4.55 ± 0.10c 7.27 ± 0.03a
MT3 27 ± 1.00ab 9.05 ± 0.05ab 80.77 ± 0.20c 90.48 ± 0.48b 5.71 ± 0.10b 3.81 ± 0.20b
LSD0.05 4.908 0.277 6.56 1.928 0.5194 1.752
Level of sig. * * * * ** **
P value 0.0413 (*) 0.0387 (*) 0.0234 (*) 0.0152 (*) 0.0011(**) 0.0060 (**)

Notes: The data are presented as the mean ± SD. Different superscript letters in the same column indicate statistically significant differences between
groups. MT-1: Methyl testosterone in Farm 1 (Agro-3 Fish Hatchery and Culture Farm, Trishal), MT-2: Methyl testosterone in Farm 2 (Fishtech Hatchery
Ltd, Tarakanda, and MT-3: Methyl testosterone in Farm 3 (Sharnalata Agro Fisheries Ltd, Fulbaria).
*p < 0.05.
**p < 0.01.

(a) (b) (c) (d)

F I G U R E 1 Photographs of the gonads of Nile tilapia: male, female, and intersex gonads at 0% pine pollen (PP) (a), 0.5% PP (b), 1.0% PP (c), and
1.5% PP (d). Scale bar = 50 μM

hormone does not have any harmful effect on fish flesh after halting the
treatment of tilapia fry, the effects of the hormone or its byproducts
on major organs (kidney, liver, pancreas, and gills), metabolic profile,
and nucleic acids are indecisive. Synthetic MT-treated fish have been
found to show a significant increase in plasma creatine phosphokinase
activities and DNA fragmentation in the liver tissue (Hasheesh et al.,
2011) and alter enzymatic metabolic pathways (Carla et al., 2020). In
the environment, steroids are either biodegraded or mineralized. How-
ever, due to unforeseen consequences, steroids that escape from the
tilapia hatchery into the natural water may change the dynamics of the
environment (Sudtida et al., 2021). This may have far-reaching effects
and may reverse the gender of certain natural populations, thereby
changing the gender dynamics of the population, producing more males
than females, which may lead to recruitment failure in the long run
(Megbowon, 2011). It has also been observed that MT can accumu-
late in pond sediments and can be detected in the soil within nearly
3 months after stopping treatment. Governments in many countries
have put strict restrictions on the use of synthetic steroids and

380
F I G U R E 2 Microscopic view of the male, female, and intersex gonads of Nile tilapia with (a) Optimized pine pollen (1.0%), (b) 17-α-methyl
testosterone in Farm 1 (MT-1), (c) 17-α-methyl testosterone in Farm 2 (MT-2), and (d) 17-α-methyl testosterone in Farm 3 (MT-3). The arrowhead
indicates ovarian cells, and the arrow indicates testicular cells. Scale bar = 50 μM
antibiotics on food fish on ethical judgments because of the risk caused
to humans by chemical residues in food and by antibiotic resistance
being passed on to human pathogens (Megbowon & Mojekwu, 2014).
Under such circumstances, the use of alternative chemicals and/or nat-
urally available sources with bioactive analogue of sex hormones is
highly demanding.
The statistically insignificant mean length, weight, and survival of
fry treated with different concentrations of pine pollen from the con-
trol group indicate that pine pollen may not have an adverse effect on
diet. However, a significant difference was observed in the sex ratio
analysis, which indicated that 1.0% pine pollen produced the high-
est percentage of males (92.50 ± 0.51%). The other two treatments
also produced significantly higher male proportions than the untreated
pine pollen group, which suggests that pine pollen can be an effective
natural phytosteroid to be used in sex reversal in fish.
Phytochemicals have been found to show potential anabolizing and
androgenic (or estrogenic) effects. It has been reported that T. terrestris
can increase testosterone levels and induce sex reversal in fish (Gharaei
et al., 2020; Kavitha & Subramanian, 2011). The growth performance
of tilapia was not different from that of mangosteen leaf extract at
concentrations of 0, 1, 2.5, and 5 g/L for 7 days and 0, 1, 2, and 3 g/L
for 28 days (Khakong et al., 2011). The aqueous leaf extract of B. alba
(ALEB) was used to observe the effect on growth performance of tilapia
fry by Ghosal et al. (2015) at different concentrations (0.0, 0.05, 0.10,
and 0.15 g/L) for 28 days. However, they found no significant effect
of ALEB on the growth performance of tilapia fry. The extract from
P. abulaeformis Carr. pollen has recently been used in Clariasgariepi-
nus, which showed slight but significantly inferior androgenic effects
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AZIZ ET AL .
but superior anabolic effects compared to both methyltestosterone
and testosterone (Adenigba et al., 2017). The growth performances
(in terms of weight gain and specific growth rate) with optimized pine
pollen in the present study showed significant variations when com-
pared to those with two of the three MT-treatment groups (MT-1,
MT-2) during the 90-day period of rearing but not with the other
(MT-3). Since the nutrients remain the same as those in other environ-
mental settings, the effective intake and digestion of feed treated with
optimized pine pollen is expected. The MT-3 group may have a similar
situation in this group, or other factors (water quality and/or inherent
factors) may have been considered.
Various studies have found that the survival of fry varies with syn-
thetic steroids and natural sterols. Survival (%) was found to be higher
in a number of studies where natural phytosteroids were used, for
example, 93.8% in tilapia fry treated with 0.15 g/L aqueous leaf extract
of B. alba for 28 days (Ghosal et al., 2015) and 92.33% in tilapia
fry treated with 0.05 g/L Mucuna pruriens for 30 days (Mukherjee
et al., 2015). With 17-MT, Das et al. (2010) obtained 92.45%, 95.41%,
93.75%, and 89% survival rates of tilapia fry with doses of 15, 30, 60,
and 120 mg/kg feed, respectively. Soltan et al. (2013) obtained 82-
67-84% survival in Nile tilapia fry after 4 weeks of 17-MT treatment
(60 mg/kg feed). While using Diethylstilbestrol (DES) for feminization
of Nile tilapia, Sultana et al. (2020) obtained 34.33%, 30.66%, and 42%
survival with 100, 200, and 300 mg/kg feed treated for a month. We
observed sufficiently high survival (> 80%) on all three farms using
MT, ranging from 80.77 to 90.77. However, the high survival (>90%)
with all concentrations of pine pollen used in the present study dur-
ing the 28-day period and no mortalities (for the optimized group)

AZIZ ET AL .
thereafter until euthanasia reveals that pine pollen could be a worthy
dietary substitute for tilapia monosex production.
The present study provided evidence that pine pollen-treated feed
resulted in a high proportion of masculinization. The percentage of
male tilapia was observed to be 92.51%, higher than that in any of
the MT-treated groups. However, various studies have shown that a
higher percentage of masculinization could also be obtained with MT
(Kohinoor et al. 2003; López et al., 2007). In contrast, with the 0.1 g/L
aqueous leaf extract of B. alba, Ghosal et al. (2015) obtained the highest
value of 70.3% for male tilapia. Cek et al. (2007) obtained the highest of
80% males with 0.15 g/L gokshura (Tribulusterrestris) on Poeceliareticu-
lata. With powdered velvet bean (5 g/kg feed), M. pruriens, Mukherjee
et al. (2015) obtained a significantly higher (p < 0.05) percentage of
male tilapia (73.33%). Withania somnifera root powder was used for
monosex (93.73%) tilapia production by De and Chakraborty (2016).
The plant material W. somnifera root powder was also reported to show
the presence of phytochemicals such as tannins, saponins, steroids, ter-
penoids, and flavonoids, which might be associated with its androgenic
property. Pine pollen also has important bioactive components, includ-
ing DHEA, amino acids, brassinosteroids, gibberellins, vitamins B and
E, and testosterone, for which this plant extract can be very effective in
supporting health and the immune system in addition to effective sex
reversal.
All three concentrations of pine pollen showed significantly higher
proportions of males and lower proportions of females compared to
the control group. The minimum percentage of 5% of females is com-
parable to the situation in the major commercial tilapia hatcheries
in Bangladesh, where best hatchery practices and optimal MT doses
still produce at least 5% females. The results obtained from the three
selected farms treated with MT also showed a similar trend (5%–7%
females, Table 2). In this study, 0.5% pine pollen produced a significantly
higher percentage of females (23.63). A slight increase (1.5%) from the
optimal concentration also produced a higher percentage of females
(16.52%). Using B. alba leaf extract at a concentration of 0.15 g/L, 33.2%
female tilapia was obtained (Ghosal et al., 2015). Reduced masculiniza-
tion and paradoxical feminization may be observed in tilapia treated
with high concentrations of synthetic steroids (Beardmore et al., 2001;
Bombardlli et al., 2007).
Undifferentiated gonads (intersex) are not uncommon in Nile
tilapia. On one of the farms (MT-2), the intersex proportion was
found to be higher (7.27%) than that on the other two farms and in
the optimized pine pollen-treated group. Factors that may account
for incomplete differentiation of gonads may vary. The presence
of endocrine disrupting chemicals was hypothesized for the occur-
rence of intersex tilapia in a contaminated river in Taiwan (Sun &
Tsai, 2009). Adeogun et al. (2016) studied the gonadal pathology of
127 cichlids from the Ogun River, Nigeria, receiving effluents from
anthropogenic activities and found 20.68% intersex fish, with the
highest degree of intersex (74%) recorded in O. niloticus and the
lowest (6%) in Tilapia guineensis. Male fish displayed higher inter-
sex (67%) than female fish (33%). They also claimed that intersex
displayed in exposed fish may have resulted from untreated efflu-
ents with endocrine disrupting characteristics discharged into this
26938847, 2022, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/aff2.59 by Turkey Cochrane Evidence Aid, Wiley Online Library on [10/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
381
river. Mateen and Ahmed (2007) found fewer intersex (2%) treated
with synthetic androgen 17-α MT at various doses (50, 60, 70, and
80 mg/kg of feed). Higher proportions of intersex tilapia (12.4) were
also observed with the phytochemical ALEB when used at 0.05 g/L by
Ghosal et al. (2015). Our study with optimized pine pollen, however,
revealed far fewer (%) intersex individuals that did not significantly
differ from those obtained from two farms practicing MT treatment
(2.50 ± 0.05, p > 0.05) and without any apparent abnormalities in gonad
structure.
5 CONCLUSION
The present study explores the efficacy of pine pollen extract at an
optimized dose (1%) incorporated into feed that could significantly
increase the sex ratio toward males in Nile tilapia without any signif-
icant adverse effects on growth performance or survival. Knowledge
of the anabolic and androgenic effects of this plant extract on tilapia, a
widely distributed global aquaculture species, is new. Laboratory anal-
ysis shows that pine pollens are ’micronutrient storerooms’ containing
concentrated growth stimulants and support hormonal balance and
the immune system, apart from the androgenic functions. However, as
there seemed significant variations regarding the efficacy of different
phytochemicals for the production of the all-male fish population and
as there are potential anabolizing and virilizing effects of such plant
extracts, we need clear documentation of this plant extract regard-
ing its mode of action, androgenic-anabolizing, and immunological
efficacies for which future work is warranted.
ACKNOWLEDGEMENTS
This work was supported by a grant from the Ministry of Science
& Technology, Government of the People’s Republic of Bangladesh,
Dhaka—1000, Bangladesh.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
Md. Abdul Aziz: Methodology. Mst. Momtaz Mostary: Methodology;
writing original draft. Israt Jahan Sume: Data curation; Method-
ology. Md. Helal Uddin: Investigation; reviewing & editing. Mohd
Golam Quader Khan: Data curation; formal analysis; writing; review-
ing & editing. Md. Samsul Alam: Investigation; validation; visualization.
M. Sadiqul Islam: Conceptualization; supervision; writing; reviewing &
editing.
DATA AVAILABILITY STATEMENT
The authors confirm that the data supporting the findings of this study
are available within the article [and/or] its supplementary materials.
ORCID
Mohd Golam Quader Khan https://orcid.org/0000-0002-9421-5269
M. Sadiqul Islam https://orcid.org/0000-0001-9357-4254

382
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