Effects of thyroxine immersion on Sterlet sturgeon (Acipenser ruthenus) embryos and larvae: Variations in thyroid hormone levels during development

Journal Pre-proof Effects of thyroxine immersion on Sterlet sturgeon (Acipenser ruthenus) embryos and larvae: Variations in thyroid hormone levels during development

Soheil Alinezhad, Hamed Abdollahpour, Naghmeh Jafari, Bahram Falahatkar PII:

S0044-8486(19)30839-7

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734745

Reference:

AQUA 734745

To appear in:

aquaculture

Received date:

9 April 2019

Revised date:

17 September 2019

Accepted date:

14 November 2019

Please cite this article as: S. Alinezhad, H. Abdollahpour, N. Jafari, et al., Effects of thyroxine immersion on Sterlet sturgeon (Acipenser ruthenus) embryos and larvae: Variations in thyroid hormone levels during development, aquaculture (2019), https://doi.org/10.1016/j.aquaculture.2019.734745

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© 2019 Published by Elsevier.

Journal Pre-proof Effects of thyroxine immersion on Sterlet sturgeon (Acipenser ruthenus) embryos and larvae: variations in thyroid hormone levels during development

Soheil Alinezhad a,*, Hamed Abdollahpour b, Naghmeh Jafari b, Bahram Falahatkar b

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Institute of Agricultural Education and Extension, Agricultural Research, Education and

Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara,

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b

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Extension Organization (AREEO), Tehran, Iran

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Guilan, Iran

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Running head title: Effect of thyroxine on Sterlet sturgeon

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*Correspondence: S. Alinezhad, Institute of Higher Technical and Vocational Education,

Tehran, Iran

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Agricultural Jihad, Agricultural Research Education and Extension Organization (AREEO),

E-mail: [email protected]

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Journal Pre-proof Abstract This study was planned to investigate the effects of immersion of Sterlet sturgeon (Acipenser ruthenus) eggs by thyroxine hormone (T4) on the embryonic survival rate (ESR), hatching rate (HR), larval survival rate (LSR), and thyroid hormone levels. Hence, oocytes of six females were pooled, fertilized with pooled semen of three males and 45 min after fertilization process, the eggs were placed 120 min in four batches that were immersed in T4 solutions as follows: D1 (the

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control treatment: T4-free, hatchery water), D2 (0.01 mg L-1 of T4), D3 (0.05 mg L-1 of T4), D4

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(0.1 mg L-1 of T4), and D5 (1 mg L-1 of T4). Next, the eggs were transferred to the incubator.

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The ESR was recorded 6 hours after fertilization while HR, and LSR were measured at 1 day

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post hatching. In addition, the levels of thyroid hormones were measured using the competitive enzyme immunoassay method three hours and three days after fertilization and at the five-day

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pre-larval stage. The highest ESR was obtained following D5 treatment (P < 0.05). Besides, the

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highest and lowest HR were observed in D5 and D1 treatments, respectively (P < 0.05). The D5 treatment resulted in the highest pre-larval SR. During all sampling times, the D5 treatment

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resulted in the highest level of thyroid hormones among the other treatments (P < 0.05).

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However, the T3:T4 ratio was significant only within the three days of fertilization in D1 treatment (P < 0.05). In general, it can be concluded that the immersion of Sterlet sturgeon eggs in T4 improved the ESR, hatching, and LSR of this species, resulting in a promising future for the aquaculture of Sterlet sturgeon and other sturgeon species. Keywords: Thyroxine, Immersion, Acipenser ruthenus, Hatching rate, Larvae survival.

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Journal Pre-proof 1. Introduction Although larva-farming techniques have been developed regularly in recent years, the early mortality rate especially in sturgeon species, which are seriously endangered, is still a major issue in hatcheries (Gisbert and Williot, 2002; Williot et al., 2009). Improving the nutrition and growth conditions of larvae increases the survival rate in intensive aquaculture (Gisbert and Williot, 1997; Abdollahpour et al., 2019). In addition, the use of hormones is a novel way of

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improving larval development (Lam and Sharma, 1985; Ayson and Lam, 1993; Kang and Chang,

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2004; Abdollahpour et al., 2018). Thyroid hormones (THs) serve as effective growth promoters

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in aquaculture (Lam 1994; Brown and Kim, 1995). These hormones are remarkably effective in

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controlling the metamorphosis and survival rates of different fish species (Contrera et al., 2016; Schnitzler et al., 2016). Research suggests that three hormone groups, including THs, growth

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(Higgs et al., 1982; Lam, 1994).

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hormone, and steroids can improve fish survival and growth, both individually and collectively

Triiodothyronine (T3) and thyroxine (T4) have been identified in the eggs of various fish

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species, with their role in controlling larval growth and improving survival rate (Kobuke et al.,

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1987; Tagawa and Hirano, 1987). However, the excessive external administration of THs has negative effects on growth, skin, bones, and muscles (Eales, 1979). Contradictory findings have been reported about THs due to variations in doses and times (Eales, 1979; Higgs et al., 1982). Some suggestions have been made concerning the use of T3 and T4 as growth promoters in aquaculture systems (Higgs et al., 1982; Castillo et al., 2013). THs are essential for fetal and larval growth through penetrating into the eggs via the adult fish (Power et al., 2001; Abdollahpour et al., 2018). Exposing fishes to external THs will increase the concentration of pigments in tissues along with the hatching and growth rate, swim bladder inflation, muscle

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Journal Pre-proof development, metamorphosis, and larval metabolic capacity (Kang and Chang, 2004; Walpita et al., 2007; Landines et al., 2010; Abdollahpour et al., 2018). According to recent studies, THs can affect the reproduction of fish and growth, and survival rate of larvae in aquaculture systems (Khalil et al., 2011; Castillo et al., 2013; Abdollahpour et al., 2018). Sturgeon species have focused by the aquaculturists due to their high adaptability potent to environmental conditions as well as rapid growth, insignificant need for oxygen, and potential

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for growth in different systems (Falahatkar and Poursaeid, 2013). Proper broodstock

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management before and after spawning can result in the production of high-quality eggs and

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larvae required for recharging reserves or aqua farming (Akhavan et al., 2016; Adollahpour et

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al., 2018). Being one of the smallest sturgeons, Sterlet sturgeon (Acipenser ruthenus) is an important species among the sturgeon in the Central Europe. This species lives in freshwater

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rivers and matures earlier than the other sturgeon species (Szczepkowski and Kolman, 2011), so

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it is used in the production of caviar in many countries. This fish provides high-quality meat and is considered as a valuable aquarium fish species. Owing its substantial importance to its

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hybridization with Beluga (Huso huso) and other sturgeon species.

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Given the economic value of sturgeon species, the important role of T4 in eggs, and their positive effects on fetal and larval performance, this study was carried out to examine the effects of egg immersion at different T4 levels on the hatching and survival rates of Sterlet sturgeon.

2. Materials and methods 2.1.

Sampling and culture conditions

This study was conducted at Shahid Dr. Beheshti Sturgeon Fish Propagation and Rearing Complex (Rasht, Guilan, Iran). In this study, firstly, the bioassays of 30 females’ broodstock

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Journal Pre-proof were carried out and oocyte maturity assessed using the germinal vesicle position (Akhavan et al., 2016). Secondly, 10 female fish samples, being in the same oocyte maturity phase and having almost equal weights, were selected. The fish average weight was 1215.2 ± 30.5 g (mean ± SE) and its average total length was 64.2 ± 2.2 cm. The fish were held in a fiberglass tank with an approximate capacity of 1065 liters. During the farming phase, filtered river water was provided as flow-through system. The average flow rate

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of the fish tank was 14 L min-1. In addition, water temperature and dissolved oxygen were

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measured and recorded daily. The average water temperature was 9 ± 0.3 °C and the mean

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dissolved oxygen was 7.5 ± 0.2 mg L-1 throughout the study. In addition, lighting conditions

Induced spawning of fish

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2.2.

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were provided naturally.

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Before the induction of fish, feeding was ceased. When the water temperature rose to the propagation temperature (16-17 °C) and the sexual maturity and spawning preparation of the

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broodstock were ensured, the fish were propagated. Fish were immersed in the 400 ppm of the

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clove powder extract and then hormonal induction was induced in two steps in female fish through the injection of LHRH-a2 at 12-hour intervals; in addition, spermiation was induced in male fish in one step (concurrent with the second injection of the female fish) through injecting a 2.5 μg kg body weight-1 into the back muscle) (Ghiasi et al., 2017). Twenty hours after the first injection, the female fish were checked individually every hour and were propagated after observing oocyte ovulation. Propagation was conducted through minimally invasive surgical technique (Pourasadi et al., 2009) administering anesthesia using 400 ppm of the clove powder extract.

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Journal Pre-proof Twelve hours after the injection of males, fish were examined with their sperms extracted through their genital pores using a 50 mL syringe (Ghiasi et al., 2017). Before using the sperms, their activities were checked and the high-quality ones were selected (Fauvel et al., 2010). At the end, the oocytes of 6 females and sperms of 3 males used for fertilization purpose.

2.3.

The T4 administration method

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Firstly, the T4 hormone (Merck, Darmstadt, Germany) was dissolved into 1 mL of ethanol (Razi,

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Khuzestan, Iran). The eggs of 6 females were mixed with the sperms of 3 males for fertilization

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process (Pourhosein Sarameh et al., 2019). After removing adhesion using clay solution and 45

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min after fertilization process, 5 g of non-adhesive eggs (~ 348 eggs per liter) were put separately in solutions containing different concentrations of T4 in 1-liter containers to examine the effects

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of hormone on embryo survival rate (ESR), hatching rate (HR) and larvae survival rate (LSR) of

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Sterlet sturgeon larvae. The eggs were immersed into five doses of T4 based on the following treatments of D1 (without T4, hatchery water), D2 (0.01 mg L-1 of T4), D3 (0.05 mg L-1 of T4),

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D4 (0.1 mg L-1 of T4), and D5 (1 mg L-1 of T4). Each treatment had 4 replication. The dosage

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and duration of exposure to T4 were determined based on the previous studies (Urbinati et al., 2008; Castillo et al., 2013). The fertilized eggs (45 min after fertilization process; in this stage previteline space was formed) were then exposed to the hormone for 120 min during the hardening (the egg animal region is divided by the first groove) (Dettlaff et al., 1993). They were next gently washed with hatchery water and transferred to special baskets in Yushchenko incubator. During the incubation, the ESR, HR, and LSR were calculated. In addition, following the hatching, larval weight and length were also measured via a digital balance and scale in 1 mg and 1 mm accuracy, respectively. In order to measure the THs in the tissues, 300 to 600 mg

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Journal Pre-proof fertilized eggs in three hours, embryo three days after fertilization and at the larval stage, 5 days after fertilization were sampled and stored at -70 °C till measuring the hormones.

2.4.

The extraction and measurement of THs

The tissues were homogenized in 5 volumes of ice-cold phosphate buffer (PBS 0.1 M, pH 7.2) by a homogenizer (Ultra turrax T25, Janke and Kunkel Labortechnik, Staufen, Germany). Next,

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300 μL of the homogenized tissue was transferred to new tubes and homogenized in 6 mL

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ethanol cooled in ice. The homogenized tissue was shaken horizontally for 10 min at 4 °C. Next,

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the samples were centrifuged at 4 °C for 15 min at 1600 g, with the upper solution transferred to

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new tubes. Tissue homogenization was performed twice using methanol, yet 2 mL of methanol cooled in ice was used at the second time instead of 6 mL of ethanol. After each centrifugation

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phase, the upper solution was removed and transferred to a new tube. At the next stage, the tubes

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were stored for 24 hours at 56 °C to allow for the evaporation of methanol. In addition, 5 μL of methanol, 200 μL of chloroform, and 50 μL of the barbital buffer (0.1 M; pH = 8.6) were added

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to the dried extract left in the tubes were shaken horizontally for 10 min. Next, the upper layer,

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which contained the thyroid hormones, was transferred to clean tubes and dehydrated at 56 °C in 3 hours. The dehydrated extracts were then dissolved in one milliliter of the barbital buffer, which contained 0.1% of gelatin, and were stored at -70 °C (Abdollahpour et al., 2018). The plasma levels of T3 and T4 were measured using laboratory diagnostic kits in accordance with Monobind’s instructions (Lake Forest, CA, USA). In addition, the absorption levels of the samples read using an ELISA reader (Epoch 2, Microplate Spectrophotometer, Vermont, USA). Finally, the hormone concentrations were calculated as ng g-1 based on the standard curve (Abdollahpour et al., 2018).

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2.5.

Statistical analysis

The data analysis was carried out by the SPSS software (version 16, Chicago, USA). All percentage data were converted into arcsin √(x/100). After assessing the homogeneity of variances using the Levene’s test and testing the normal distribution of the data using the Kolmogorov-Smirnov test, the results were analyzed. The multivariate general linear model was

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used to analyze the variations of THs. In this model, five hormonal treatments and three

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sampling times (3 post-fertilization hours, 3 post-fertilization days, and the 5-day pre-larval

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stage) were the constants, and the levels of T3 and T4 hormones as well as the T3:T4 ratio were

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the variables. To determine the significance level, the Duncan’s test was used as a post-hoc test,

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with the significant statistical difference approved at the P < 0.05 level.

3.1.

Incubation indices

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3. Results

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There was a significant difference among the treatments in ESR, HR, and LSR (P < 0.05; Table

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1). The highest ESR and HR were indicated in D5 treatment compared to the other groups (P < 0.05). This difference was also significant among different treatments, except for treatments D2 and D3. The highest and lowest ESR were obtained in D5 (83.44%) and D1 (58.06%), respectively. D5 and D1 treatments also resulted in the highest (80.03%) and lowest (27.66%) HR, respectively. In treatment D5, the larvae had a higher SR (P < 0.05). The LSR in D2 to D4 treatments did not differ significantly from that of the control treatment, but the difference was significant for treatment D5 with the mean SR of 82.40%. However, there was no significant difference among treatments in the larval weight and length (P > 0.05).

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3.2.

Hormonal parameters

T3 and T4 levels as well as their ratio (T3:T4) were measured using different hormonal treatments (Table 2). T4 level was significantly affected by the treatments at all three sampling times (P < 0.05). In the 3 h post-fertilization, the highest T4 level was 275.72 ± 6.52 ng g-1 for treatment D5 (P < 0.05) and the lowest T4 level was 182.46 ± 37.58 ng g-1 for treatment D1 (P <

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0.05). There was a significant difference among all treatments during this sampling period (P <

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0.05). Moreover, the T4 level of the eggs increased significantly in the D5 treatment 3 days

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following fertilization in contrast to the other treatments (P < 0.05). The highest level of T4 was observed in treatment D5 at 199.88 ± 19.59 ng g-1, and the lowest level of T4 was seen in

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treatment D1 at 145.37 ± 8.95 ng g-1 (P < 0.05). The T4 level in 1-day larvae in treatment D5

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was significantly different from the other treatments. Treatments D5 and D1 differed

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significantly with each other and resulted in the highest (193.26 ± 5.53 ng g-1) and lowest (144.75 ± 13.74 ng g-1) levels of T4, respectively (P < 0.05).

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The level of T3, three hours after fertilization, varied significantly among the treatments, while it

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was the highest level in treatment D5 among the other treatments (P < 0.05; Table 2). In addition, the highest (246.89 ± 2.37 ng g-1) and lowest (113.18 ± 15.01 ng g-1) T3 levels were observed in treatments D5 and D1, respectively. T3 level of the eggs increased significantly in treatment D5 three days after fertilization, compared with treatments D1, D2, and D4 (P < 0.05). The highest and lowest T3 levels in three-day eggs were observed in treatments D5 (181.52 ± 9.20 ng g-1) and D1 (113.18 ± 15.01 ng g-1), respectively (P < 0.05). T3 level in 1-day larvae in treatment D5 was significantly different from treatments D1 and D2. Moreover, the highest (157.78 ± 10.02 ng

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Journal Pre-proof g-1) and lowest (118.15 ± 17.14 ng g-1) T3 levels were observed in treatments D5 and D1, respectively (P < 0.05). The T3:T4 ratio, in egg tissues 3 hours after fertilization, was significantly higher in treatment D5 than the treatments D1 and D2. In contrast, treatment D5 yielded the lowest T3:T4 ratio (1.12 ± 0.12) (P < 0.05). This ratio increased significantly in treatment D1 compared with treatment D3 in egg tissues 3 hours after fertilization (P < 0.05). The T3:T4 ratio in the 1-day larvae did

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not differ significantly among experimental treatments (P > 0.05).

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The effects of time on T4 and T3 concentrations as well as the T3:T4 ratios have been illustrated

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in Table 3. The highest T4 level (238.03 ± 37.85 ng g-1) was observed three hours after

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fertilization, whereas the lowest T4 level (161.02 ± 24.53 ng g-1) was obtained three days after fertilization. The difference in hormone concentrations three hours after fertilization and during

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the two other times was significant (P < 0.05). In addition, there was no interaction between the

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treatments and the sampling times (P > 0.05). T3 concentration was significantly higher three hours after fertilization than the other times (P < 0.05). The highest (179.28 ± 16.33 ng g-1) and

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lowest (136.65 ± 18.74 ng g-1) concentrations were obtained three hours after fertilization and

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one-day larvae, respectively. In addition, there was a significant difference in the hormone ratio three hours after fertilization and the other times (P < 0.05). The hormone ratios were 1.37 ± 0.25 (the highest amount) and 1.12 ± 0.17 (the lowest amount) three hours after fertilization and oneday larvae, respectively.

4. Discussion The literature review identified no study was conducted on the effects of immersion the eggs in T4 on sturgeon embryo and larvae. The present experiment, however, showed that T4 improved

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Journal Pre-proof the ESR and HR successfully, thereby improving the larval viability of Sterlet sturgeon. Moreover, 1 mg L-1 treatment produced the best response among all treatments. According to previous studies, the presence of T3 and T4 in the eggs improves initial growth as well as larval and egg survival and it may because of that following fertilization process of the eggs, the hormone concentrations are declined as yolk consumption and THs are involved in newly hatched embryo performance (Kimura et al., 1992; Ayson and Lam, 1993; Kang and

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Chang, 2004; Castillo et al., 2013; Contrera et al., 2016). In confirmation to the previous

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research and based on our findings, the THs levels in our experiment indicated a tendency

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decrease during Sterlet development and it may show that THs is involved in Sterlet embryo and

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larval viability. In general, the administration of THs to female sturgeons and increase transferring of these hormones from blood to target tissues increase the quality of eggs and

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larvae (Tagawa and Hirano, 1987; Tagawa and Brown, 2001; Abdollahpour et al., 2018, 2019).

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It was also found out that the ESR in the D5 treatment was higher than the other treatments. This finding is in line with the results of another study on the close correlation between gonad

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maturation and thyroid levels in rainbow trout (Cyr and Eales, 1988).

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Previously, the positive effects of THs have been reported on hatching rate of common carp (Cyprinus carpio) (Lam and Sharma, 1985). Moreover, it has been confirmed that THs improve fetal and larval growth or hatching efficiency in some fish species (Urbinati et al., 2008; Castillo et al., 2013; Abdollahpour et al., 2018). The different results in the researches on the initial embryo survival-improving impact of THs are possibly because of variation in the trial design, method of T4 administration, T4 dosages, differences between the species and the developmental stage at which hormones are increased (Nacario 1983; Ayson and Lam 1993; Mylonas et al.

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Journal Pre-proof 1994). The effects of THs on SR of eggs in T4 treatment can also be attributed to increased resistance to fungal and parasitic infections (Harris and Bird, 2000; Lam et al., 2005; Tort, 2011). The considerable effects of THs on newly hatched larvae have been reported by several researchers. For instance, T4 accelerates larval growth, and improve the SR in fish species (Lam, 1980; Lam and Sharma, 1985; Kang and Chang, 2004). The findings from a study on the effects of 1 to 10 μg g BW-1 in T4 treatment on Nile tilapia (Oreochromis niloticus) larvae indicated

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faster development of digestive system and larval viability (Khalil et al., 2011). In addition, the

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effects of injection of 20 μg T3 kg BW-1 on rockfish (Sebastes schlegeli) were indicated an

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increase in T3 levels of plasma, eggs and larvae of this species, but no impact was reported on

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T4 concentration. The growth rate of fish receiving the T3 was also higher than that of the control group. The larval survival rate was higher in fish receiving T3 injections than the other

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treatments (Kang and Chang, 2004). In another experiment, 10 and 20 μg kg BW-1 T3 was

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injected into matrinxa (Brycon amazonicus), indicating the hormonal treatment did not affect the hatching time. However, upon an increase in level of THs, larval deformities decreased and

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larvae had immersed in hormone gained more weight (Urbinati et al., 2008). In addition, thyroid

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stimulating hormone improved larval growth and reproductive efficiency of alligator gar (Atractosteus spatula) and spotted gar (Lepisosteus oculatus) (Castillo et al., 2013). The results of this experiment showed that an increase in T3 and T4 concentrations at the embryonic development stage improved the HR and reproductive efficiency of fish. Another study on Mozambique tilapia (Oreochromis mossambicus) eggs reported the lack of a specific mechanism for controlling the inflow of T3 into the oocytes. Furthermore, it was stated that the T3 concentration increased in the oocytes while its concentration was increased in the culture medium, yet it did not reach the saturation point (Tagawa and Brown, 2001). The findings from a

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Journal Pre-proof study on rainbow trout eggs indicated that incubation using a T3-rich ovarian fluid (10 to 100 μg mL-1 doses) resulted in a significant increase in the T3 concentration in oocytes. However, the oocytes exposed to a T3-free follicular liquid showed lower T3 concentrations (Raine and Leatherland, 2003). In addition, the weekly injection of T3 into rockfish broodstock within a month resulted in considerable increase in T3 concentrations in rockfish eggs (Kang and Chang, 2004).

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In the higher vertebrates, THs play various roles, such as physiologic growth, metabolic

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regulation, and hemostasis (Avivi et al., 2014; De Souza et al., 2017). In the lower vertebrates,

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other tasks, such as metamorphosis stimulation in amphibians are attributed to THs, which is

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probably the best advantage of THs (Orton and Tyler, 2015; Buchholz, 2017). THs in the higher mammals exert immediate biological effects on their life (Orton and Tyler, 2015; Buchholz,

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2017). These hormones play a vital role in the growth of fish in fetal and larval phases, yet their

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long-term effects on embryonic development and larval growth have yet to be determined (Sullivan et al., 1989; Kang and Chang, 2004).

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Even though, the dose of hormone and the technique of administration may be crucial parameters

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affecting the results (Moon et al. 1994; Power et al. 2001). In this study, we did not detect any deformity in larvae of Sterlet sturgeon, despite some researchers have stated that using THs directly to fish embryos or larvae could resulted in malformations in larvae and reduced survival rate, maybe as an outcome of an overdose (Nacario, 1983; Mendoza et al. 2002; Walpita et al. 2007). In current study, the significant elevation of THs levels in the eggs from the hormoneadministrated, as contrasted with controls, indicated the transfer of T4 from the hatchery water into the eggs, as has been reported in other fish species (Nacario, 1983; Hey et al., 1996; Castillo et al., 2013). Given the results of the present experiment, increase in hatching and larval SRs of

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Journal Pre-proof Sterlet sturgeon is because of THs increase in tissues. Also, THs levels in treatment indicated the steady decreasing of the concentrations of THs in each treatment in subsequent developmental stages which could have been attributed to the role of 5'-monodeiodinase. It is concluded that THs penetrate into the eggs through the egg walls and are stored in the eggs within 120 minutes of exposure, given the strong correlation between the concentration of hormones in the culture medium and high T3 and T4 concentrations in different eggs and larval tissues in Sterlet

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sturgeon.

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Conclusion

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Fish farmers need high-quality eggs and offspring. Hence, researchers analyzed variety of methods in order to increase the quality and fertility of eggs to find a proper respond to this

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growing demand appropriately. The results of the past research and the present study are

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indicative of the vital role of THs in the critical stages of growth and development of Sterlet sturgeon. According to the findings from the present experiment, immersion of Sterlet sturgeon

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eggs in T4 improves the incubation efficiency of this species, thereby potentially reducing

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commercial farming costs. Furthermore, improvements in the HR and SR of Sterlet sturgeon were quite obvious in the final results of the present experiment, where the high dose of T4 (1 mg L-1) was the most effective. Finally the immersion of eggs in T4 improves the larval survival rate of Sterlet sturgeon.

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Journal Pre-proof Abdollahpour, H., Falahatkar, B., Efatpanah, I., Meknatkhah, B., Van Der Kraak, G., 2019. Hormonal and physiological changes in Sterlet sturgeon Acipenser ruthenus treated with thyroxine. Aquaculture 507, 293-300. Abdollahpour, H., Falahatkar, B., Efatpanah, I., Meknatkhah, B., Van Der Kraak, G., 2018. Influence of thyroxine on spawning performance and larval development of Sterlet sturgeon

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Akhavan, S.R., Salati, A.P., Falahatkar, B., Jalali, S.A.H., 2016. Changes of vitellogenin and lipase in captive Sterlet sturgeon Acipenser ruthenus females during previtellogenesis to

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Hormones and Evolution Academic Press, London, pp. 342-436. Falahatkar, B., Poursaeid, S., 2013. Stress responses of great sturgeon Huso huso subjected to husbandry stressors. Aquac. Int. 21, 947–959. Fauvel, C., Suquet, M., Cosson, J., 2010. Evaluation of fish sperm quality. J. Appl. Ichthyol. 26, 636-643. Ghiasi, S., Falahatkar, B., Arslan, M., Dabrowski, K., 2017. Physiological changes and reproductive performance of Sterlet sturgeon (Acipenser ruthenus) injected with thiamine. Anim. Reprod. Sci. 178, 23-30. 16

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Khalil, N.A., Allah, H.M.K., Mousa, M.A., 2011. The effect of maternal thyroxine injection on growth, survival and development of the digestive system of Nile tilapia, (Oreochromis niloticus), larvae. Adv. Biosci. Biotechnol. 2, 320-329. Kimura, R., Tagawa, M., Tanaka, M., Hirano, T., 1992. Developmental changes in tissue thyroid hormone levels of red sea bream, Pagrus major. Nippon Suisan Gakkaishi 58, 975. Kobuke, L., Specker, J.L., Bern, H.A., 1987. Thyroxine content in eggs and larvae of coho salmo, Oncorhynchus kisutch. J. Exp. Zool. 242, 89 - 94.

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Journal Pre-proof Lam, T.J., 1994. Hormones & egg/larvae quality in fish. J. World Aquacult. Soc. 25, 2-12. Lam, T.J., Sharma, R., 1985. Effects of salinity and thyroxine on larval survival, growth and development in the carp, Cyprinus carpio. Aquaculture 44, 201-212. Lam, T.J., 1980. Thyroxine enhances larval development and survival in Sarotherodon (Tilapia) mossambicus Ruppell. Aquaculture 21, 287-291. Lam, T.J., Sharma, R., 1985. Effects of salinity and thyroxine on larval survival, growth and

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Journal Pre-proof Pourasadi, M., Falahatkar, B., Azari Takami, G., 2009. Minimally invasive surgical technique for egg collection from the Persian sturgeon, Acipenser persicus. Aquac. Int. 17, 317-321. Pourhosein Sarameh, S., Bahri, A.H., Falahatkar, B., Yarmohammadi, M., Salarzadeh, A.R., 2019. The effect of fish and rapeseed oils on growth performance, egg fatty acid composition and offspring quality of sterlet sturgeon (Acipenser ruthenus). Aquacult. Nutr. 25, 543-554.

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development and metamorphosis in Cyprinodon variegatus. Aquat. Toxicol. 181, 1-10. Sullivan C., Bernard, M., Hara, A., Dickhoff, W., 1989. Thyroid hormones in trout reproduction:

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enhancement of GnRH analogue and partially purified salmon gonadotropin- induced ovarian maturation in vivo and in vitro. J. Exp. Zool. 250, 188–195. Szczepkowski, M., Kolman, R., 2011. A simple method for collecting sturgeon eggs using a catheter. Arch. Pol. Fish. 19, 123-128. Tagawa, M., Brown, C.L., 2001. Entry of thyroid hormones into tilapia oocytes. Comp. Biochem. Physiol. 129B, 605-611.

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Journal Pre-proof Tagawa, M., Hirano, T., 1987. Presence of thyroxine in eggs and changes in its content during early development of chum salmon, (Oncorhynchus keta). Gen. Comp. Endocrinol. 68, 129135. Tort, L., 2011. Stress and immune modulation in fish. Dev. Comp. Immunol. 35, 1366-1375. Urbinati, E.C., Vasques, L.H., Senhorini, J.A., Souza, V.L., Gonçalves, F.D., 2008. Larval

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larval rearing of captive endangered Atlantic sturgeon Acipenser sturio. Endanger. Species

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Journal Pre-proof Table 1. The effects of different levels of thyroxine on fertilization, hatching, and survival rates (mean ± SE) of Sterlet sturgeon (Acipenser ruthenus). Thyroxine levels (mg L-1) Incubation indices

Control

0.01

0.05

0.1

1

58.06 ± 0.77d

67.39 ± 1.01c

33.96 ± 1.21c

70.98 ± 0.88b

83.44 ± 1.59a

Hatching rate (%)

27.66 ± 2.32 d

51.51 ± 2.28 c

50.50 ± 2.74c

59.5 ± 1.75b

80.03 ± 2.28a

Survival rate at 1 dph (%)

41.67 ± 7.90b

42.39 ± 3.62b

48.36 ± 10.59b

55.11 ± 1.57b

82.40 ± 4.85a

Larval weight at 1 dph (mg)

12.32 ± 0.82

12.32 ± 0.18

12.32 ± 0.91

12.32 ± 0.56

12.32 ± 0.69

Larval length at 1 dph (mm)

10.90 ± 0.20

10.75 ± 0.20

10.90 ± 0.45

10.95 ± 0.40

Embryonic

survival

rate

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(%)

10.90 ± 0.30

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dph: day post hatch. Data indicated by different letters in each row are statistically significant (P <

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0.05).

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Table 2. The effects of different levels of thyroxine on T4, T3 and T3:T4 (mean ± SE) of Sterlet sturgeon (Acipenser ruthenus).

s

time

T3:T4

0.05

0. 1

1

3 hpf

182.46 ± 37.58d

227.17 ± 4.68cd

246.11 ± 12.66bc

258.68 ± 26.80b

275.72 ± 6.52a

3 dpf

145.37 ± 8.95b

140.40 ± 8.89b

150.20 ± 7.49b

169.23 ± 3.11b

199.88 ± 19.59a

5 dpl

144.75 ± 13.74b

154.64 ± 7.49b

155.12 ± 20.87b

161.65 ± 20.17b

193.26 ± 5.53a

3 hpf

119.21 ± 13.86d

150.38 ± 8.26c

188.71 ± 8.80b

191.23 ± 15.56b

246.89 ± 2.37a

3 dpf

113.18 ± 15.01 c

132.19 ± 10.24bc

163.08 ± 11.97b

138.38 ± 14.78c

181.52 ± 9.20a

5 dpl

118.15 ± 17.14b

128.61 ± 5.37b

141.71 ± 16.6ab

137.04 ± 21.03ab

157.78 ± 10.02a

3 hpf

1.56 ± 0.47a

1.51 ± 0.11a

1.31 ± 0.12ab

1.35 ± 0.13ab

1.12 ± 0.12b

3 dpf

1.30 ± 0.22a

1.06 ± 0.03ab

0.93 ± 0.09b

1.23 ± 0.10ab

1.10 ± 0.09ab

5 dpl

1.20 ± 0.02

1.10 ± 0.15

1.20 ± 0.24

1.23 ± 0.22

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T3 (ng g-1)

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T4 (ng g-1)

Thyroxine levels (mg L-1)

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Sampling

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Parameter

1.24 ± 0.19

hpf: hours post fertilization, dpf: days post fertilization, dpl: days pre-larvae. Data indicated by different letters in each row are statistically significant (P < 0.05).

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Table 3. The effects of sampling time on the levels of T4, T3, and the T3:T4 ratio (mean ± SE) in Sterlet

T3 (ng g-1)

179.28 ± 16.33a

T3:T4

1.37 ± 0.25a

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238.03 ± 37.85a

161.02 ± 24.53b

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T4 (ng g-1)

3 days after fertilization

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3 hours after fertilization

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Parameters

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sturgeon (Acipenser ruthenus).

5 days pre-larvae 161.88 ± 21.32b

145.67 ± 145.67b

136.65 ± 18.74b

1.19 ± 0.14b

1.12 ± 0.17b

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Data indicated by different letters in each row are statistically significant (P < 0.05).

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Journal Pre-proof Highlights of the manuscript 

Thyroxine immersion is remarkably effective in improving hatching and survival rate of Sterlet sturgeon.



Higher T4 and T3 concentrations were obtained at 3 hours after fertilization in the highest level of T4 treatment. Higher and lower larval survival rate were recorded in D5 and D1 treatment, respectively.



Thyroxine immersion had no significant impacts on larval weight and length.

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