Comparative study on accuracy of mucosal estradiol-17β, testosterone and 11-ketotestosterone, for maturity, and cutaneous vitellogenin gene expression in goldfish (Carassius auratus)

Knowledge of

sex

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identification and

reproduction

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outputs in

fish

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still requires more details to optimize aquaculture facilities management, particularly on some

species

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, they are mature later than the male, or hermaphrodite

species

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with a

sex

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ratio managing reason, etc. (

Barkowski

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and

Haukenes

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, 2014; Budd et al., 2015; Chen et al., 20201-3). A relevant low-risk method is become an important option to determine

reproduction

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outputs based on

sex

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and

maturity

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status.

Although

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several methods have been used, so far, in

sex

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and

maturity

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identification, including ultrasonography (Moghim et al., 2002;

Kohn

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et al., 20134-5), laparoscopy (Falahatkar et al., 20116), plasma

sex

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steroid assay (Mandich et al., 2004;

Kohn

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et al., 2013; de Carvalho et al., 20175, 7-8) and gonad histology (Petochi et al., 2011; Falahatkar and Poursaeid, 20149-10), some of them are considered as invasive techniques. Among the different procedures,

blood

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sampling is usually regarded as a robust source of

reproduction

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indicator including

sex

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steroids

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and vitellogenin (

VTG

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) known as critical regulators of the

reproduction

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cycle and

sex

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indicator (Mandich et al., 2004; Orlando et al., 2007; Loke-Smith et al., 2010; Sole et al., 20167, 11-13). The

blood

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females'

maturity

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markers, estradiol-17β (E2) and

VTG

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(Garnayak et al., 2013; Reading et al., 201714-15), as well as

blood

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androgenic

steroids

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[testosterone (T) and 11-ketotestosterone (11-KT)] involved in males’ spermatogenesis and spermiation processes (Barry et al., 1990; Schulz et al., 199016-18), are commonly used in

sex

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determination (Falahatkarand and Poursaeid, 2014; de Carvalho et al., 20178, 10).

However

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, they cannot be recommended as a robust tool owing to the stressful process sampling, which may interfere with

fish

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welfare status. In recent years, the

skin

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mucosal layer has been seen several functions which are related to the

fish

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immunity system (Subramanian et al., 2008; Roosta et al., 2014; Sanahuja et al., 2019; Abolfathi et al., 202019-22), environmental disruptors (Meucci and Arukwe, 2005; Fernandez-Alacid et al., 2018; Bolluch et al., 202023-25) and somewhat

reproduction

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diagnostic characteristics (

Barkowski

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and

Haukenes

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, 20141).

Mucus

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, a preferred method by supporting environmental safety and conservation of

species

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, can be utilized as a surrogate of

blood

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, due to the fact that it prevents

fish

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sacrificing, minimizes the physiological stress or can be collected even if repeated samplings are required (Schultz et al., 2005; Ekman et al., 2015; Dzul-Caamal et al., 2016; Oliveria et al., 201726-29), that possesses similar components.

Skin

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mucosal tissue indeed contains a lower range of

reproduction

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substances comprising 11-KT (Schultz et al., 2005;

Barkowski

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and

Haukenes

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, 20141, 26) and

VTG

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(Moncaut et al., 2003; Pottinger et al., 2005;

Kohn

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et al., 2013;

Barkowski

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and

Haukenes

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, 20141, 5, 30-31) rather than

blood

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plasma;

therefore

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, their contents may be less effective to identify the

sex

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ratio or

maturity

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stages (

Kohn

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et al., 20135). In

this

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study, goldfish (Carassius auratus), a well-known animal model, has been selected. It is frequently used in

fish

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reproduction

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physiology, endocrinology, ecology and immunology. Goldfish is a seasonal spawner

species

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, which could be easily maintained under natural and laboratory conditions to monitor its performance (Popesku et al., 2008; Nelson and Habibi, 2010; Gong et al., 2016; Blanco et al., 2018; Thompson et al., 201832-36). The main target of

this

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study was to corroborate maturational progress, plus

sex

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identification through

mucus

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sex

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steroids

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included E2, T, 11-KT. There are a number of physio-biological components in

skin

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mucus

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that proved our hypothesis about

mucus

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secretion in

reproduction

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physiology. In that regard,

this

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study was designed to explore the accuracy of

sex

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steroids

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along with Ca and

VTG

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as a non-invasive diagnostic way.

In addition

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, the circulation of

sex

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steroids

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in

fish

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mucus

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, detection of E2 and T, can lead to new knowledge in

fish

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skin

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indices. The

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vtg

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big

gene expression was

also

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determined in different

reproduction

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related-tissues.