Comparative study on accuracy of mucosal estradiol-17β, testosterone and 11-ketotestosterone

This

study

was designed

a

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as a

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comprehensive non-invasive procedure towards

sex

identification

and

maturity

status that might be applied for other teleost species as well. Herein, E2,

T

, 11

-KT

,

VTG

and Ca of

mucus

were tracked, clarified and

then

compared to

plasma

, and gonad analyses in goldfish. In the present

study

, mucosal androgens considering for

sex

identification

, and even contributing

in

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to

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males

' reproductive status as an indicator.

T

had an acceptable trend

resulted

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that resulted

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in

sex

identification

, its accuracy was 89%, at the same time, had the lowest

level

(33%) of

identification

efficacy in

maturity

status. Regarding different roles of

T

playing in spermatogenesis (Schulz et al., 2010; Basak et al., 2016), synthesizing of 11

-KT

in spermiation evaluation

besides

males

' sexual behaviour hormone (Barry et al., 1990), apparently can track in

mucus

in order to its

further

potential. Measurement of

T

levels

in

water

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the water

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maintaining Dentex dentex and in

plasma

, related to the number of

females

stocked in the tank (Pavlidis et al., 2004).

Nevertheless

,

T

level

in

females

’

skin

mucus

has been currently lower than

males

. Florida gar (Lepisosteus

platyrhincus

Correct your spelling

platyrrhines

), group-synchronous teleost spawned,

sex

steroids in

plasma

showed an upward trend as it comes to reproduction season (Orlando et al., 2007). Already there is a group-synchronous pattern herein, but the border of stages is apparently distinguishable across both goldfish genders. Only 11

-KT

of

plasma

,

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seemingly hit a peak in spring over upcoming warm. Indeed both mucosal androgens were fairly stable in pre-reproduction and reproduction seasons. Schultz et al., (2005) reported 13-fold higher for a range of mucosal 11

-KT

in

males

than

females

of Koi (Cyprinus carpio), which was not far from our results (

mucus

content was 7-ply compared with

plasma

). What is more, considering Barkowski and Haukenes (2014) indicating a direct relationship between 11

-KT

of

mucus

and

plasma

, strengthening of mucosal hormone is hands-on.

Sex

identification

in white bass (Morone

chysops

Correct your spelling

chops

) was correctly determined by 90% in the following, and a low error rate by 11

-KT

in

mucus

had been seen

as a result

.

Similarly

, the 11

-KT

accuracy has been observed at

maturity

stage detection (83%) and

sex

identification

(89%). Inadequate knowledge on fish

skin

mucus

components leads to hardly find out of how much

sex

steroids exist within,

in particular

E2 and

T

,

However

, there are several studies in mammals that proved the diffusion procedure and tissues' origin of

mucus

(Friedl et al., 2013; Wang & Shi 2017; Lock et al., 2018). The mucosal layer might play passive and active roles during the diffusion process relied on the shape and size of

skin

and

mucus

(Dünnhaupt et al., 2015), and fish

sex

hormones differentiation over seasonal and

sex

alteration might directly affect

skin

structure as a consequence (Pickering & Pottinger, 1985; Pottinger, 1987). Based on our

study

, E2

levels

in

mucus

primarily were high at initial and late-vitellogenesis, even rather than

plasma

. In

further

, at vitellogenesis, protein

levels

of

plasma

were on top more than

mucus

. The reason why these two matrices have a significant difference.

Although

there is no data about fish mucosal E2, investigation of

estrogens

encoding a protein in

mucus

and epithelial cells have proved

hypothesis

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the hypothesis

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of controlling

mucus

might be aligned with

estrogens

, a pivotal hormone in reproduction (Massart et al., 2004). In

this

survey, appearing of E2 in male’s

mucus

would not be debatable since it seems rational predicted the

mucus

VTG

level

or

vtg

gene

expression

in the

skin

. The ova-testis hypothesis and environmental

estrogens

disruptors’ effects were failed by monitoring testis histological slides. There is a potent tendency for inducing a gender reverse in cyprinids (Saoshiro et al., 2013; Hassell et al., 2016), and

somales'

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sales

behaviour would probably

affectfemales

Correct your spelling

affect females

or vice versa. Apart from androgens, E2 plays a critical role in mammals’ spermatogenesis process (Carreau & Galeraud-Denis, 2008), or even estrogen can take a burden as mediator testicular function and spermatogenesis in teleosts (Miura et al., 1999; Kobayashi et al., 2011; Laing et al., 2018). Pottinger et al., (2005) reported that

plasma

E2 could not be a suitable discriminator of

sex

in sea trout (Salmo trutta) and Atlantic salmon (Salmo salar). Even though, a high

level

of E2 in

plasma

of mature

females

is indicated in Atlantic cod (Gadus morhua) (Dahle et al., 2003), goldfish (Kagawa et al., 1983; Kobayashi et al., 1988) and Florida gar (Orlando et al., 2007). Turning primary-oocytes into mature ones through E2 affected ovary, or controlling the

gene

expression

in different tissues like the

liver

(Marlatt et al., 2010) and E2 circulation in the blood all are key stimulator to

VTG

synthesis in the

liver

. Normally,

VTG

is synthesized and detectable only in matured

females

(Denslow et al., 1999; Tian et al., 2009). Yet, the hypothesis of physiological interaction in

mucus

, the

first

line of body components, and

then

direct interface with the peripheral aqueous medium (Fletcher et al., 1997; Sanahuja et al., 2019) leading to

combination

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a combination

the combination

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of environmental

estrogens

. In the present

study

,

mucus

VTG

was identically at a low

level

compared to

plasma

, but it was apparently detectable and acceptable.

Low

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The low

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expression

level

of the

vtg

in

males

'

skin

or/and even the

level

of E2 in

males

' body confirm their

VTG

secretion.

VTG

can be transmitted from

plasma

to

mucus

by a diffusion mechanism or another regulated mechanism unknown (Kishida et al., 1992), and since

VTG

is the yolk protein produced in the

liver

by

estrogens

activity; as a consequence, it is transferred to the ovary for oocyte as a final destination (Meucci & Arukwe 2005; Tian et al., 2009; Reading et al., 2017). High

levels

of

vtg

gene

expression

in the

liver

,

therefore

, would be expected inconsistent with our results.

Similarly

,

vtg

Correct article usage

the vtg

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gene

expression

of ovaries was more than

liver

in white cloud mountain minnow (Tanichthys

albonubes

Correct your spelling

bonuses

) (Wang et al., 2010) and sterlet sturgeon (Acipenser ruthenus) (Akhavan et al., 2016).

Sex

identification

has been more distinguished by

skin

mucus

Ca (73%) and

VTG

(61%) than E2 (44%). Certainly, because blood Ca is an indirect tool for detection of

VTG

during

maturity

progress, Ca content can be assayed as a biomarker of

estrogens

recirculation in

females

at once

VTG

levels

(Svoboda et al., 2001; Linares-Casenave et al., 2003; Stahl et al., 2009).

Although

the Ca

level

of

plasma

was higher than

mucus

, there is

sustainable

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a sustainable

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amount of Ca observed in the

mucus

particularly during vitellogenesis.