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Journal of Animal Science - Animal Genetics

Technical note: Production of tetraploid sturgeons1

 

This article in JAS

  1. Vol. 93 No. 8, p. 3759-3764
     
    Received: Mar 17, 2015
    Accepted: May 19, 2015
    Published: July 10, 2015


    2 Corresponding author(s): ilebeda@frov.jcu.cz
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doi:10.2527/jas.2015-9094
  1. I. Lebeda 2 and
  2. M. Flajshans
  1. University of South Bohemia, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic

Abstract

Studies and practical application of androgenesis and gynogenesis in sturgeon are significantly hindered by strong influence of ploidy restoration treatment on survivability of progeny; therefore, developed method of production of tetraploid broodstock and, consequently, use of their diploid gametes might help to avoid ploidy restoration treatment. In the present study, for the first time was developed a protocol for tetraploidy induction in 2 model sturgeon species, sterlet (Acipenser ruthenus) and Siberian sturgeon (Acipenser baerii). A high efficiency of treatment was achieved by optimization of heat shock using a temperature of 37°C for 2 min timed between the end of female pronuclei formation and the beginning of pronuclei migration, that is, 0.8 to 1.0 τo (duration of 1 mitotic cycle during the period of synchronous cleavage division). Fertilized eggs developed in tetraploid larvae, up to 31 (89.6% in control) and 34% (70.9% in control) in sterlet and Siberian sturgeon, respectively. Most of the tetraploid larvae exhibited body malformations; as a result, consequent large scale study revealed high larval mortality, which drastically decreased after 2 mo of age. Consequent comparison of BW, length, and malformation rate and mortality between diploid and tetraploid progeny of sterlet did not reveal significant differences in fitness of diploid and tetraploid juveniles at 9 and 11 mo of age. The present study can be considered the first step towards improving the androgenesis methods of conservation of endangered sturgeons as well as understanding the sturgeon sex determination system through induction of mitotic gynogenesis.



INTRODUCTION

Altogether, the 27 extant sturgeon species are one of the most highly imperiled groups of taxa, with a worldwide reduction in abundance and distribution (Birstein et al., 1997; Rosenthal et al., 2006). This depletion as well as the uniqueness of sturgeon phylogeny has forced a great deal of research in sturgeon genetics and aquaculture. Tetraploid sturgeons, particularly their diploid gametes, possess great interest for further ploidy manipulation or production of gynogenotes and androgenotes without the necessity for rediploidization, as was done in rainbow trout by Thorgaard et al. (1990). The diploid gametes of tetraploid individuals open a wide field for production of polyploids, as was done in loach Misgurnus anguillicaudatus (Arai et al., 1993) or rainbow trout Oncorhynchus mykiss (Chourrout and Nakayama, 1987). In addition, diploid sperm of endangered sturgeon represent a great interest for conservationists because of a possibility of restoring the population from cryopreserved diploid sperm by interspecific androgenesis (Grunina et al., 2011). Particular attention has been devoted to a similar treatment used in mitotic gynogenesis induction as one of the main methods to understand the sex determination system in sturgeon species. The goal of the present study is to develop protocols for production of tetraploid sturgeon for further application in studies of a sturgeon sex determination system by induction of mitotic gynogenesis, investigation of influence of ploidy level on gametes physiology, and improving the androgenesis method for conservation of endangered sturgeons.


MATERIAL AND METHODS

In the first part of this study, we carried out optimization of a protocol of temperature shock for mitotic tetraploidization in sterlet and Siberian sturgeon. For the treatment, we applied temperatures commonly used for triploidization treatment and for fusion of sperm nuclei during dispermic androgenesis in sturgeon, namely 34 to 37°C (Recoubratsky et al., 1996; Fopp-Bayat et al., 2007; Grunina et al., 2011). In addition, we optimized the time span from the moment of extrusion of the second polar body (0.6 τo [duration of 1 mitotic cycle during the period of synchronous cleavage division] at 16°C) to prophase of the first mitotic division (1.6 τo at 16°C; Dettlaff et al., 1993). Duration of 1 τo is approximately 63 min in sterlet and 59 min in Siberian sturgeon at a cultivation temperature of 16°C according to Dettlaff et al. (1993) and Gisbert and Williot (2002). In the next part of the study, we used the obtained optimal parameters of heat shock to produce tetraploid sterlet for further rearing of tetraploid adults.

We obtained the fish for this experiment from the Genetic Fisheries Centre, Faculty of Fisheries and Protection of Waters in Vodňany, Czech Republic. The spermiation in males was induced according to a previously published method (Linhart et al., 2000). We induced ovulation in females by hormonal stimulation with a carp pituitary suspension and then collected the ovulated eggs by microsurgical incisions in the oviducts, as described previously by Gela et al. (2008).

For fertilization, we added 20 mL of water (15°C) to 125 μL of sperm, or an equivalent amount of diluted sperm (625 μL), and immediately added this suspension to 4 g of eggs, according to Gela et al. (2008). The eggs were fertilized for 2 min with gentle stirring at 16°C, and we then distributed them into 3 petri dishes. After the eggs were attached, the petri dishes were immersed in a thermostabilized tank and maintained at 16°C until we performed the heat shock treatment.

We performed the heat shock treatment by immersing the eggs that had been incubated at 16°C into hot water. Optimization was done in the time range from telophase II stage to the beginning of the pronuclei encounter, that is, 0.4 and 1.6 τo, respectively (Dettlaff et al., 1993). The tetraploidization optimization setup is described in Table 1. After heat shock, we immediately placed the petri dishes back into the incubation system at 16°C and incubated them until hatching. To prevent fungal infection, dead eggs were removed after 2 d of incubation when the neural tube was clearly visible.


View Full Table | Close Full ViewTable 1.

Tested parameters of heat shock treatment for tetraploidization in sterlet and Siberian sturgeon. Duration of 1 mitotic cycle during the period of synchronous cleavage division (τo) is approximately 63 min for sterlet and approximately 59 min for Siberian sturgeon at 16°C according to Dettlaff et al. (1993) and Gisbert and Williot (2002)

 
Species Tempera-ture,°C Duration, min Starting time, min Starting time, τo
Sterlet 34 2 56 0.88
74 1.75
87 1.38
99 1.57
3 56 0.88
74 1.75
87 1.38
99 1.57
37 2 50 0.79
56 0.88
74 1.75
87 1.38
99 1.57
4 50 0.79
56 0.88
74 1.75
87 1.38
99 1.57
Siberian sturgeon 37 2 41 0.7
47 0.8
53 0.9
59 1.0
65 1.1
71 1.2
3 41 0.7
47 0.8
53 0.9
59 1.0
65 1.1
71 1.2

In order to check the success of the tetraploidization treatment, we analyzed the ploidy level of up to 10 hatched larvae from each petri dish, which corresponded with 30 samples per group. We minced a tissue sample of the caudal part of the fin and incubated it in Nuclei Extraction Buffer (CyStain DNA 2step; Partec GmbH, Münster, Germany). Nuclei in separated and permeabilized cells were stained with fluorescent DNA dye, 4′,6-diamidino-2-phenylindole. A Cube 8 flow cytometer (Partec GmbH) was used to estimate the relative DNA content per cell. The untreated diploid Acipenser ruthenus larvae were used as a reference. At least 2,000 nuclei were analyzed in each sample with the flow-through rate of 0.5 to 1.0 μL/s.

The mass production of tetraploid sterlet was done using optimal parameters of heat shock from previous optimization, namely 37°C for 2 min at 56 min after fertilization. For this, 100 g of eggs (approximately 7,500 eggs) was fertilized with 2.5 mL of sperm. After fertilization, eggs were washed with tap water and transferred in clay suspension at 16°C for desticking. For heat shock, eggs were collected into containers with a mesh bottom and transferred to preheated 37°C clay suspension for 2 min. After heat shock, eggs were incubated for 30 min in clay suspension at 16°C and then divided into 2 incubation jars. After hatching, larvae were volumetrically counted and cultivated in a 100-L tank with constant flow of hatchery water. At 2 mo of age, juvenile fish were counted and transferred to a closed recirculation system. Ploidy levels of putative tetraploids were analyzed by flow cytometry of blood samples on Cube 8 flow cytometer at 9 mo of age, following the protocol of Linhart et al. (2006).


RESULTS AND DISCUSSION

The idea behind this method is based on the application of treatment (physical or chemical shock) during the zygote’s first mitotic division. There are reports of similar treatment being used in fish species, for example, in common carp (Cyprinus carpio; Wu et al., 1986), tench (Tinca tinca; Flajshans et al., 1993), ayu (Plecoglossus altivelis; Taniguchi et al., 1990), and Nile tilapia (Oreochromis niloticus; Peruzzi et al., 1993) and for rediploidization of androgenetic homozygotes, for example, in rainbow trout (Scheerer et al., 1986).

The application of mitotic heat shock with an intensity similar to commonly used triploidization treatment (34°C for 2 min) in sterlet resulted in a relatively high hatching rate of 70%. However, the tetraploidization efficiency was extremely low and only 1 tetraploid larva was found (in the group treated 74 min after activation for 2 min). This might be caused by a difference in target mechanisms of mitotic and meiotic shocks. In addition, it may indicate a higher depolymerization tolerance limit of the protein complexes involved in mitotic division (Harris et al., 1989; Komen and Thorgaard, 2007). Several larvae showed a ploidy level of 3n, which might be a result of retarded early development and suppression of the second meiotic division. Increasing the duration of shock to 3 min did not substantially affect the hatching rate, and it also did not increase treatment efficiency; we did not identify any tetraploid larvae.

In contrast, treatment with a higher temperature (37°C) was sufficient to produce mainly tetraploid progeny. However, eggs hatched only in groups treated for a shorter period of time (2 min) and hatching rates were substantially lower than after application of heat shock at 34°C (Fig. 1). A similar treatment was used in sturgeon to restore diploidy during androgenesis induction, such as during dispermic androgenesis induction in Siberian sturgeon (Grunina et al., 2011) and in starry sturgeon Acipenser stellatus (Recoubratsky et al., 1996). This treatment was focused on fusion of sperm nuclei after polyspermic fertilization (Grunina et al., 2011) and was applied at the beginning of the prophase stage of the first mitotic division, namely 1.4 to 1.6 τo after fertilization (Recoubratsky et al., 1996; Grunina et al., 2011).

Figure 1.
Figure 1.

Hatching rate and percentage of hatched tetraploid larvae of sterlet relative to the heat shock. Eggs were immersed in a water bath at 37°C for 2 min (duration of 1 mitotic cycle during the period of synchronous cleavage division [τo] is approximately 63 min at a cultivation temperature of 16°C according to Dettlaff et al. [1993]). Groups treated with heat shock for 4 min did not hatch. Data with the same superscript (a–dLatin for hatching rate and α,β,γGreek for percentage of tetraploid larvae) did not differ significantly at P < 0.05.

 

The highest efficiency of sterlet tetraploidization was found after application of heat shock with a temperature of 37°C for 2 min starting 56 min (0.88 τo) after the eggs were activated. This treatment resulted in 34.5 ± 4.3% hatched eggs and all the processed larvae were tetraploid (n = 25). This timing corresponded with the end of female pronuclei formation and the beginning of migration (Dettlaff et al., 1993) and can be applied for other sturgeon species after taking into account species-specific differences in duration of mitotic division related to incubation temperature. This confirmed the existing assumption that the mechanism underlying mitotic heat shock is based on suppression of normal microtubules dynamics (Chourrout, 1982).

Based on these results, we chose only a temperature of 37°C for optimization of tetraploidization treatment in Siberian sturgeon. Almost no neurulation and subsequently no hatching were found in Siberian sturgeon embryos treated with a longer duration of shock (3 min). On the contrary, a short shock (2 min) resulted in a relatively high hatching rate (up to 31.6%) in the group treated with a temperature of 37°C 59 min after activation (Fig. 2). Subsequently, ploidy analysis of hatched larvae showed high efficiency of tetraploidization; therefore, solely tetraploid progeny were produced by heat shock starting at 47, 53, and 59 min after egg activation. As a result, the highest percentage of tetraploid larvae (31.6%) was produced by heat shock with a temperature of 37°C for a duration of 2 min at 59 min (1 τo) after egg activation. This corresponded to the timing of pronuclei migration (Dettlaff et al., 1993), similar to the sterlet tetraploidization described above.

Figure 2.
Figure 2.

Hatching rates and percentage of hatched tetraploid larvae of Siberian sturgeon relative to the timing of the heat shock. Eggs were immersed in a water bath at 37°C for 2 min. (duration of 1 mitotic cycle during the period of synchronous cleavage division [τo] is approximately 59 min at a cultivation temperature of 16°C according to Gisbert and Williot [2002]). The data from groups treated with heat shock for 3 min are not presented. Data with the same superscript (a–dLatin for hatching rate and α,β,γ,δGreek for percentage of tetraploid larvae) did not differ significantly at P < 0.05.

 

The obtained hatching rates and tetraploidization efficiency were relatively high, taking into account that tetraploidy has been obtained in only a few fish species and mostly with a low efficiency (Pandian and Koteeswaran, 1998). It is likely that tetraploidization was induced more easily in sturgeon due to the plasticity of their ploidy system.

On the other hand, even though our results showed efficient tetraploid larvae production, the survival rate of larvae produced for mass tetraploid production was low, probably due to the number of malformations (Fig. 3). Tetraploidization usually has quite a low success rate and leads to the appearance of malformations in larvae development (Thorgaard et al., 1981; Chourrout and Nakayama, 1987; Nagoya et al., 1990; Sumantadinta et al., 1990; Varadaraj, 1990). This could have been due to the heat shock, which is a known source of malformations, especially given the increased temperature compared with the one used during triploidization (Fopp-Bayat et al., 2007).

Figure 3.
Figure 3.

The appearance of normal larvae of sterlet (A) and malformed tetraploid larvae in this species (B) 2 d after hatching.

 

Large-scale tetraploidy induction resulted in 600 hatched larvae (approximately 12% of eggs). Ploidy analysis of hatched larvae showed more than a half of progeny was tetraploid (20 larvae; 67%), about one-third was diploid (9 larvae; 30%), and 3.3% was triploid. After 2 mo, when the stock was transferred to recirculation system, only 47 fish survived, which corresponded with roughly 1% of treated eggs. After 2 mo of age, mortality substantially decreased; subsequently, 41 fish survived until the ninth month. Ploidy analysis of 9-mo-old putative tetraploids showed doubled content of DNA in 34.15% of the fish. This decrease of proportion of tetraploids in progeny indicated their higher mortality compared with normal fish. Nevertheless, comparison of weight and body malformation rate of tetraploid and diploid juveniles did not reveal significant difference at 9 and 11 mo of age (Table 2). Therefore, it could be assumed that tetraploidy by itself did not significantly affect development and fitness-related parameters of juveniles and that larval mortality was caused by other consequences of the heat shock.


View Full Table | Close Full ViewTable 2.

Comparison of weight and body malformation rate of tetraploid and diploid juveniles of sterlet at 9 and 11 mo of age

 
Age, mo Ploidy Number of fish (percentage) Average weight, g Malformation rate, number of malformed fish (% of malformed)
9 2n 27 (66%) 20.1 2 (7.4%)
4n 14 (34%) 21.2 1 (7.1%)
11 2n 26 (65%) 45.9 2 (7.7%)
4n 14 (35%) 47.4 1 (7.1%)

Obtained tetraploid progeny could be used for production of diploid gametes, which could be used to avoid negative consequences of ploidy restoration treatment in production of triploids, gynogenotes, and androgenotes.

 

References

Footnotes


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