Abstract:

All-male tilapia aquaculture is desirable to control unwanted breeding. Besides, male tilapia
individuals grow faster and bigger than females. Presently, most farmers use 17amethyltestosterone
(MT) to produce an all-male stock, although the hormone is associated with
human health and environmental risks. Recently, plant-based products have been reported to
induce masculinisation in fish and are considered safe nature-based alternatives to MT. The
present study utilised pine pollen (PP) to induce female-to-male sex change in Nile tilapia
(Oreochrotnis niloticus). Prior to the start of the research, there was insufficient information
on the use of PP for sex inversion, with no published data on the sex change mechanism, hence
limiting the progress in the application of the product from experimental to hatchery levels.
In this study, the optimal dietary inclusion of PP for maximum masculinisation of Nile tilapia
compared with fish of the same batch fed the same basal diet with no PP (CT; negative control)
or the same basal diet supplemented with 60 mg MT kg'! (MT; positive control). To confirm
whether the sex change was complete, fish in all treatments were fed only a basal diet for an
additional 84 days. The associated differences in the growth of the fish were also determined.
Pine pollen and MT significantly skewed the expected 50:50 (male: female) ratio towards more
male individuals (Chi-square: X2 = 54.396, df = 9, P 0.001). The 1,280 mg PP kg1 of diet
MT (89.2 ± 2.2 %), and both were
significantly higher than 50.8 ± 2.2 % in the CT treatment. In addition to masculinization,
dietary’ inclusion of 1,280 mg PP kg’1 improved fish growth, with the specific growth rate
significantly higher than fish from the MT and CT treatments (One-way’ ANOVA:
n
equally induced masculinisation (80.0 ± 2.9 % males) as
1,920, 2,560 and 3,200 mg kg-1 basal diet) from 3 to 30 days post-hatch (dph). This was
was investigated by feeding three-day-old fish graded PP levels (80, 160, 320, 640, 1,280,
0.001). An increment in F(9.20) -14.196, P the dietary levels of PP from 1,280 to 3,200
mg kg'1 further promoted the growth of the fish but did not affect masculinisation.
The mechanism underlying PP-induced sex masculinisation was investigated using all-female
Nile tilapia fed a basal diet supplemented with 1,280 mg PP kg'1 for 28 days from 3 dph, in
comparison with fish fed a basal diet incorporated with 60 mg MT kg’1 (MT treatment) or only
foxl2), changes in sex steroid profiles (T: testosterone, 11-KT: 11-ketotestosterone, and E2:
Gene expression and sex steroid
concentrations were significantly influenced by the interaction between dietary treatment and
time, with the expression changing differently over time among the treatments (RM-ANOVA:
P < 0.001). Pine pollen significantly up-regulated the expression of dmrtl and amh, while
cypl9ala and foxl2 were down-regulated. Corresponding to male sex gene up-regulation,
male-based steroids (11-KT and T) levels were also significantly amplified in both PP and MTtreated
fish. The gene expression pattern and changes in sex steroids corresponded to a higher
proportion of male individuals obtained in the MT and PP treatments (MT: 97.8 ± 1.1 % and
PP: 77.8 ± 2.9 % males), implying female-to-male sex change induction. Subsequently,
spermatogonia and spermatocytes were the dominant germ cells in the histological sections of
the gonads obtained from the PP-treated fish. At the same time, the individuals from the MT
treatment exhibited mainly spermatids and spermatozoa. In contrast, all the fish from the CT
treatment remained females, having only ovarian tissues.
This thesis confirmed that PP induces female-to-male sex change in Nile tilapia and enhances
the mechanism underlying PPiii
a basal diet (CT treatment). The expression of sex-related genes {dmrtl, amh, cypl9ala, and
fish growth. The research contributed novel information on
17p-estradiol), and gonadal histology were analysed.
induced sex change, which included disrupting the expression of sex genes and the androgenfoundation
for understanding the role of PP in masculinisation, with broad potential application
in the aquaculture industry.
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