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Study on artificial induction, growth and gamete quality of mitogynogenetic turbot Scophthalmus maximus
Aquaculture 515 (2020) 734585
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Study on artificial induction, growth and gamete quality of mitogynogenetic turbot Scophthalmus maximus
T
Zhihao Wua,b, Lijuan Wanga,b, Qiaowan Wua,b,c, Yunliang Lud, Zongcheng Songe, Jun Lia,b, Feng Youa,b,∗ a
Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China c University of Chinese Academy of Sciences, Beijing 10049, China d Qingdao Agricultural University, Qingdao 266237, China e Shenghang Aquatic Science and Technology Co. Ltd., Weihai 264200, China b
ARTICLE INFO
ABSTRACT
Keywords: Turbot Scophthalmus maximus Artificial mitogynogenesis induction Growth Sex ratio Offspring
Artificial gynogenesis induction is one of the important techniques of chromosome manipulation. The eggs are fertilized with inactivated sperm to prevent the inheritance from the paternal genome, and genome information of the offspring is totally contributed by the maternal genome. Mitogynogenesis, in which diploidization of chromosome set is performed by blocking the cleavage, is theoretically homozygous and has got more attention in breeding. In this study, we performed the mitogynogenetic diploid induction in turbot Scophthalmus maximus, one of the most important maricultural fish in China and Europe, by applying hydrostatic pressure to the eggs activated with UV-irradiated homologous sperm. The optimal inducing conditions were 36,000 erg mm-2 for UVirradiated and pressure of 65 MPa for 6 min at 15 min before the appearance of the cleavage furrow. Two mitogynogenesis stocks were obtained in 2016 and 2017. The results showed that there was no distinct difference in incubation time and morphological traits between mitogynogenetic diploid and control diploid. The survival rates of mitogynogenetic diploid sharply decreased in the first month post hatching (mph). Their total weight (tW) and total length (tL) increased rapidly from 12 to 18 mph. The mitogynogenetic diploids induced in 2016 and 2017 showed very different growth. The mean tW and tL of 10 fast-growing individuals at 18 mph were 2.78 and 1.32 times of the others in 2016 stock, while 4.50 and 1.74 times in 2017 stock, respectively. The higher male ratio and rising trend of male ratio indicated the female heterogametic ZW/ZZ genetic mechanism of sex determination in turbot. Twenty-four adults in 2017 stock survived until 24 mph. While twenty-nine adults in 2016 stock survived until 36 mph, in which 11 adults could be promoted mature. Then the qualities of gametes were evaluated and showed that both the egg and the sperm qualities of mitogynogenetic diploid were lower than those of control diploid. However, the embryo and larvae development showed no difference between mitogynogenetic diploid progeny and control diploid.
1. Introduction Artificial gynogenesis induction is one of the important techniques of chromosome manipulation. The eggs are fertilized with genetically inactivated sperm to prevent the inheritance from the paternal genome, and genome information of the offspring is totally contributed by the maternal genome (Avise, 2015; Mei and Gui, 2015; Xu et al., 2015). It has been widely utilized in selective breeding, sex control and quantitative trait locus mapping (Arai, 2002; Komen and Thorgaard, 2007). In teleost, the meiogynogenesis can be realized by inhibiting the release of
the second polar body of fertilized eggs and diploidizing the chromosome set in gynogenetic embryos. While the mitogynogenesis can be realized by blocking the early cleavage of fertilized eggs and diploidizing the chromosome set. Until now, most studies focused on artificial induction of meiogynogenesis because the survival rates were relatively high. Besides, meiogynogenetic diploids retain high level of heterozygosity as high frequency of crossover happened in the prophase of the first meiosis (Dayani et al., 2011). On the contrary, mitogynognenetic diploids were theoretically homozygous at almost all loci. It has more application potential in rapidly developing clone lines, genetic
∗ Corresponding author. Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. E-mail address: [email protected] (F. You).
https://doi.org/10.1016/j.aquaculture.2019.734585 Received 4 July 2019; Received in revised form 3 October 2019; Accepted 7 October 2019 Available online 08 October 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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mapping, production of mono-sex stocks and elimination of recessive deleterious genes (Bertotto et al., 2005). However, the survival rate of mitogynogenetic fish was low due to the body deformations or recessive alleles (Jagiello et al., 2017; Polonis et al., 2018). Therefore, artificial mitogynogenesis induction techniques have been developed only in about 20 fish species (Komen and Thorgaard, 2007), such as olive flounder Paralichthys olivaceus (Yamamoto, 1999a; Zhang et al., 2018), red sea bream Pagrus major (Kato et al., 2001), sea bass Dicentrarchus labrax (Francescon et al., 2004) and brown trout Salmo trutta (Michalik et al., 2015). Nowadays, hydrostatic pressure treatment is widely used in induction of mitogynogenesis. The moment of shock induction, pressure intensity and pressure duration are the key factors to determine whether the induction succeed. The moment of shock induction has been confirmed to be the most important factor. Different initiate time of shock induction brought extremely significant difference on induction and survival rates (Lin et al., 2015). Though mitogynogenesis induction may produce higher deformity, lower survival rates, reduced fertility and poor egg quality due to the treatment effects, deleterious recessive mutations and asynchronous embryonic development (Fopp-Bayat et al., 2017; Jagiello et al., 2018; Palti et al., 2002), the benefits of mitogynogenesis on selective breeding overwhelm the defects. The mitogynogenesis with homologous usually showed segregation of characters, such as growth, shape and color, and is an ideal method in the selection of clone lines with better growth or other commercial traits. The screening of different growth performance in gynogenesis has been conducted in cyprinid loach Misgurnus anguillicaudatus (Suzuki et al., 1985), honmoroko Gnathopogon caerulescens (Fujioka, 1998) and fancy carp Cyprinus carpio (Taniguchi et al., 1986). However, almost all such studies based on meiogynogenesis, and the utility of mitogynogenesis in breeding was reported only in a few fish, such as olive flounder (Hou et al., 2016). The studies on the growth performance and the fertility of mitogynogenesis, which are the most important factors in breeding, are still lacking. The turbot Scophthalmus maximus is an important mariculture fish in Europe and China. The annual production in China was 49.5 kilo tons, which is about 83% of the world's total output (FAO, 2018), and makes a principal contribution to the production of land-based tank cultured marine finfish in China (Lei and Liu, 2010). Therefore, the utilization of gynogenesis method in turbot breeding is valuable. Until now, some studies focused on the meiogynogenesis in turbot have been reported (Piferrer et al., 2004; Xu et al., 2008), but only one study gave a protocol of the mitogynogenesis induction in turbot, in which the eggs were activated by UV irradiated heterologous sperm (Meng et al., 2016). The study on growth, gonad development and mature performance of the mitogynogenesis in turbot is lacking, which may define its application in turbot aquaculture and breeding. In the present study, we optimized the mitogynogenesis induction conditions in turbot, and two mitogynogenetic turbot stocks were established by using the optimized condition. The early development, growth performance, sex ratio and gametes quality of the fish in the two stocks were assessed. The results will help in the induction of turbot mitogynogenesis and the breeding application.
be tested within one batch of eggs. 2.2. UV irradiation of turbot sperm The inactivation of turbot sperm was as follows. Semen was diluted to 1:10 in Ringer's solution at 0.0–2.0 °C, then the diluted semen was equally divided into chilled glass Petri dishes (13 cm in diameter), and spread 2 mm in depth. The dishes were placed in a UV box with constant shaking during UV irradiation (254 nm). The UV lamps were warmed up 10 min before onset of the irradiation. The UV exposure was set at 36,000 erg mm-2 under cool conditions (Piferrer et al., 2004). The motility of spermatozoa was checked under microscope before and after the UV irradiation by observing within 20 s. The degree of motility and the average survival rates of each sample were categorized following the reported standard at the temperature of 15.0 ± 0.5 °C. (Strussmann et al., 1994). 2.3. Artificial fertilization To perform artificial fertilization, the irradiated (5 mL diluted) or normal (0.5 mL undiluted) sperm was activated with 15–20 mL filtered seawater at 15.0 °C for 30 s, then was added to plastic beakers containing about 5000 eggs each. After gentle agitation for 30 s, 50–100 mL filtered seawater was added. Then, the eggs were rinsed carefully with seawater at 20 min post fertilization (mpf), and the floating eggs were collected. Eggs fertilized with un-inactivated turbot sperm were used as diploid control. The fertilized eggs were incubated under 15.0 ± 0.2 °C. 2.4. Mitogynogenesis induction The appropriate conditions for the production of mitogynogenetic diploid turbot were examined by varying the moment of hydrostatic pressure shock before the appearance of the cleavage furrow, or by altering the pressure or duration of hydrostatic pressure shock. Hydrostatic pressure induction was carried out in a manual hydrostatic pressure chamber. Three experiments were designed as follows: (1) to determine the optimal moment of shock induction with a single treatment of pressure shock at 65 MPa for 6 min at 5, 10, 15, 20 or 25 min before the appearance of the cleavage furrow; (2) to determine the appropriate pressure intensity of shock with a single treatment of pressure shock at 15 min before the appearance of the cleavage furrow for 6 min at 55, 60, 65, 70 or 75 MPa; (3) to determine the appropriate pressure duration of shock with a single treatment of pressure shock at 15 min before the appearance of the cleavage furrow at 65 MPa for 4, 5, 6, 7 or 8 min. The preset pressure was achieved in 15 s, and recovered to normal pressure in 30 s after shock treatment. Temperature was constantly monitored throughout the experiment. After treatment, shocked eggs were acclimated in seawater for incubation under 15.0 ± 0.2 °C. One male and one female parents were used in each experiment. All experiments were replicated five times using egg batches derived from five different females, and the males were randomly collected from the male stocks containing more than 80 individuals. Fertilization and hatching rates were determined by examining ∼ 200 and 100 floating eggs each experimental group, respectively. Survival rates of larvae indicated the rates of successfully induced larvae. Fertilization rate (%) = buoyant eggs at blastula stage/initial buoyant eggs after induction × 100%; Hatching rate (%) = normal larvae at 12 h after hatching/fertilized eggs × 100%; Survival rate (%) = fertilization rate × hatching rate × 100%
2. Materials and methods 2.1. Brood stock management and gamete collection Turbot brood stocks were cultured in the fish farm of Shenghang Aquatic Science and Technology Co. Ltd., Weihai, Shandong, China under controlled conditions (photoperiod 14 h light: 10 h dark; temperature, 14.0 ± 1.0 °C). The semen was collected from mature male by gently pressuring their abdomens and was stored in 5 mL EP tubes on ice until used for fertilization in 30 min. Semen contaminated with water or urine was discarded. The eggs were stripped from each female, and stored in a wet box under dark condition. The eggs were divided into parts, ∼5000 each before fertilization so that all parameters could
2.5. Assessment of ploidy level The ploidy of control and treatment groups was determined by cellular DNA content and haploid syndrome observation. Flow cytometric analysis with a PARTEC cell counter analyzer CCA-II (PARTEC, Germany) was performed to detect average cellular DNA contents of 2
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Fig. 1. The flow cytometric analysis of control diploid (A), haploid (B) and mitogynogenetic diploid (C) larvae in turbot Scophthalmus maximus.
hatched larvae. Thirty hatched larvae were sampled and prepared in each group (Luckenbach et al., 2004; You et al., 2001). Control diploid larvae were used as diploid standard for the calibration of the cytometer. Similar to other fish, the turbot haploids produced in treatment groups were morphologically abnormal with big heads, short tails or almost tailless. The embryos hardly hatched and died before or soon after hatching, and hence were easily identified by observation.
2.8. Statistical analysis Data from control diploid and mitogynogenesis induction groups were analyzed with the SPSS package for Windows (Version 15.0, SPSS, Chicago, IL). Data are shown as mean ± S.D. Distributions were examined for departures from normality by the Kolmogorov-Smirnov test and the homogeneity of variances was verified by the Levene's test. Significant differences were determined using One-way ANOVA tests followed by Duncan's multiple comparison tests at the probability level of 0.05.
2.6. Growth and sex ratio of mitogynogenetic diploids To assess the growth performance of mitogynogenetic turbot, according to the results of induction conditions, two mitogynogenesis stocks were induced in 2016 and 2017, respectively. The control diploids were also conducted. About 200 mL eggs acquired from 3 - 4 mature female turbot were used for the production of mitogynogenetic diploids. The eggs were fertilized with UV irradiated sperms from 3 - 4 mature male turbot, and then shocked under the optimal parameters. Then eggs were incubated in net cages under 15.0 ± 0.2 °C. The hatched larvae were reared in indoor tanks with flow-through sea water using the protocol for turbot developed by Person-Le et al. (1991) at a temperature of 18.0–21.0 °C, and fed with rotifer, artemia and commercial dry feed (salinity 28–30, pH 7.8–8.2, dissolved oxygen > 6 mg/L, water exchange rate 10% for pre-larvae, 100% for post-larvae and > 800% for juveniles and adults). The culture conditions, such as water parameters, density and feed strategy, were the same as control diploids. From 3 months post hatching (mph) to 24 mph, 30 individuals were randomly sampled from the control diploid and mitogynogenesis stocks every 3 or 6 months, respectively. After the fish were euthanized using MS-222 (60 mg/L, Sigma-Aldrich, Spain), the total length (tL) and total weight (tW) of each fish were measured. The sex of each individual was evaluated with the morphology observation of gonad by using the light through the abdomen from 6 mph to 18 mph. Gonads from five fish for each sex at 6 mph were dissected out, and the sex was confirmed with paraffin section (Ma et al., 2018).
3. Results 3.1. Sperm inactivation UV irradiation caused reduction in the survival of turbot sperm. Duration of sperm motility also declined slightly. After activation by sea water, the motility duration of UV irradiated sperm was about 20 min. The eggs could be fertilized by irradiated sperm. All the embryos exhibited haploid syndrome, and hardly hatched. The ploidy was also confirmed by flow cytometric analysis (Fig. 1). The rarely hatched haploid individual died soon after hatching. 3.2. Optimal parameters of pressure treatment The optimal parameters of pressure treatment were evaluated mainly based on the survival rate. The fertilization and hatching rates in control diploid groups ranged 80.0–98.0% and 60.1–89.7%, respectively. The treatment groups, which the fertilization rates lower than 80.0% or the hatching rates lower than 60.0%, were excluded. The time of the appearance of cleavage furrow in fertilized eggs was around 95–110 mpf under 15.0 ± 0.2 °C, but only the fertilized eggs with appearance of cleavage furrow varied from 97 - 102 mpf were used. Five batches of eggs were applied in each experiment. The fertilization rates of haploid and mitogynogenetic diploid groups were slightly lower than those of control diploid groups due to the inactivation treatment of sperm. A peak of survival rate was obtained when the shock started at 15 min before the appearance of the cleavage furrow, which was significantly higher than those obtained from shocks applied at 5 and 25 min before the appearance of the cleavage furrow (P < 0.05, Fig. 2A). Then some other factors such as shock pressure and shock duration were detected. At 65 MPa, pressure shocks of 6 min brought higher survival rates, and therefore gave the higher production of mitogynogenetic diploid (Fig. 2B and C). In terms of mitogynogenetic diploid production, the optimal conditions were 15 min before the appearance of the cleavage furrow under hydrostatic pressure shock at 65 MPa for 6 min with UV-inactivated turbot sperm.
2.7. Quality assessing of mitogynogenetic turbot gametes The mitogynogenetic turbots over 33 mph were promoted maturity by adjusting the light period and water temperature with nutrition (Imsland et al., 1997a). The male and female gametes were collected from mature fish by gently pressuring their abdomens. Semen from 3 males and eggs from 1 female were obtained. The semen was stored in 5 mL EP tubes on ice until required in 10 min. The eggs were stored at wet box under dark condition. The gamete quality was evaluated according to Valdebenito et al. (2013). The artificial fertilization procedure was the same as the control diploid described in section 2.3. The motility, survival rate and survival time of sperms, morphological traits of eggs, and fertilization and hatching rates of fertilized eggs were estimated for three replicates. The male and female gametes collected from control diploid mature fish from the same origin of mitogynogenetic turbot were also used as the control.
3.3. Production and survival of mitogynogenetic diploid Two mitogynogenetic diploid stocks were induced with the optimal protocol of pressure shock, at 15 min before the appearance of the 3
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Fig. 2. Effects of timing, intensity and duration of hydrostatic pressure shock on the relative fertilization rates and relative survival rates of haploid (n) and mitogynogenetic diploid turbot. Eggs activated with UV-irradiated sperm were shocked at 5–25 min (A) before the appearance of the cleavage furrow at 55–75 MPa (B) for 4–8 min (C); n = 5. The column indicates the relative fertilization rate, and the line indicates the relative survival rate. Data were shown as mean ± S.D.; Different letter indicates significant difference (P < 0.05), the same below. Table 1 The relative fertilization and hatching rates of mitogynogenetic diploid stocks. Stock
Fertilization rate (%)
Hatching rate (%)
2016 2017
71.9 93.3
6.5 6.1
cleavage furrow with 65 MPa shock of 6 min. The relative fertilization and hatching rates in the treatment stocks were shown in Table 1. It took about 120 h for the mitogynogenetic diploids to hatch under 15 °C as well as control diploids. There was no distinct difference in morphological traits of embryos among treatment and control stocks (Fig. 3). The hatching rates of treatment stocks were much lower than those of control stocks. All embryos in haploid stocks showed obvious haploid syndrome. Only a few malformed surviving haploids hatched but died quickly, which ensured the purity of mitogynogenetic diploids (Fig. 3). The ploidy of each stock was also tested by flow cytometry, which presented the acquirement of 100% mitogynogenetic diploid. Compared with control diploid, high mortality occurred during the periods of the first feeding (3 days post hatching, dph) and appearance of swim bladder (about 12 dph) in mitogynogenetic diploid. The survival rates of mitogynogenetic diploid sharply decreased in the first month after hatching, and also obviously decreased at the second month after hatching due to the mortality at metamorphosis period. Fish hardly died after 3 mph (Fig. 4).
Fig. 4. The survival rates of 2 mitogynogenetic diploid (2016 and 2017) and 2 control diploid stocks (2016-2n and 2017-2n).
3.4. Growth performance of mitogynogenetic diploid The growth of 2 mitogynogenetic diploid stocks was showed in Fig. 5. The tW and tL increased rapidly from 12 to 18 mph. Although the 2 stocks showed similar growth trend in the tested period, there were very different growth performance. The mean tW of 10 fastgrowing individuals at 18 mph was 2.78 times of the others induced in 2016, while 4.50 times of the 18 mph individuals induced in 2017. The mean tL of the 10 fast-growing individuals at 18 mph was 1.32 times of the others induced in 2016, while 1.74 times of the 18 mph individuals induced in 2017. Finally, twenty-nine and twenty-four fish respectively survived at 36 and 24 mph in 2016 and 2017 stocks. Both the tW and tL
Fig. 3. The development of control diploid (A), haploid (B) and mitogynogenetic diploid (C) turbot. Scale bar = 0.25 mm. 4
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Fig. 5. The total weight (A, B) and total length (C, D) of 2 mitogynogenetic diploid (2016 and 2017) and 2 control diploid stocks (2016-2n and 2017-2n).
in mitogynogenetic diploid stocks were higher than those in control diploid stocks. The larger S.D. demonstrated larger variation of growth within mitogynogenetic diploid stocks (Fig. 5). 3.5. Sex ratio of mitogynogenetic diploid The sex of each individual was evaluated with the morphology observation of turbot gonad by using the light through the abdomen from 6 mph to 18 mph (Fig. 6). The sex ratios of more than 30 randomly selected fish at 6 mph, and all the fish after 9 mph were checked. The < 60% male ratio was only observed at 6 mph in 2016 and 20162n stocks. A rising trend of male ratio was found in all the stocks, and the male ratios after 9 mph significantly deviated from the 1:1 ratio with a χ2-test (Fig. 7).
Fig. 7. The sex ratios of 2 mitogynogenetic diploid (2016 and 2017) and 2 control diploid stocks (2016-2n and 2017-2n).
3.6. Gametes quality of mitogynogenetic diploid
activated sperms kept high activity (Grade III - IV) for 16.2 ± 3.4 min (Table 2). The unfertilized eggs were buoyant, transparent, spherical in shape, and 1 mm in average diameter with a single oil globule. The first filial generation (F1) stocks were acquired. The fertilization and hatching rates of 2 F1 stocks ranged from 50.0 - 59.1% and 38.0–42.6%, respectively. Another F1 stock showed low fertilization rate due to the
The males which semen could be drawn and the females which the eggs could be stripped were considered to be mature. Nine males and 2 females in 2016 stock were promoted mature on 36 mph. The quality of sperms from three matured males and eggs from one matured female were observed and analyzed. The sperm motility of matured males was around Grade III. About 78.3 ± 2.9% sperms were activated. The
Fig. 6. The distinguishment of turbot sex using the light through the abdomen. A1-A4, male; B1-B4, female; the dotted line indicates the gonad. A1, B1, scale bar = 2 cm; A2, A3, B2, B3, scale bar = 0.5 cm; A4, B4, scale bar = 20 μm. 5
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Table 2 The sperm motility, survival rate and survival time in mitogynogenetic diploid stocks. Replicates
Motility
Survival rate (%)
Survival time (min)
1 2 3 Mean
II-III III-IV III-IV -
80.0 75.0 80.0 78.3 ± 2.9
15.0 13.5 20.0 16.2 ± 3.4
Table 3 The fertilization and hatching rates of mitogynogenetic diploid progenies. Replicates
Fertilization rate (%)
Hatching rate (%)
1 2
59.1 50.0
42.6 38.0
2019), a phenomenon being called leaky gynogenesis according to Janko et al. (2007). Therefore, to avoid unknown effects of heterologous sperm on the offspring, the homologous sperm was utilized in this study. Under the optimized sperm inactivation conditions, 100% gynogenetic haploid could be acquired. The optimal mitogynogenesis induction protocol has also been established in our study. Among the three parameters (timing, intensity and duration) of mitogynogenesis induction with pressure shock treatment, the timing is most critical because it is more species specific than the other two parameters and is usually narrowed down to a range of a very few minutes (Francescon et al., 2004; Komen and Thorgaard, 2007). In the appropriate condition, the spindle was disassembled by the treatment and then resembled in its pretreatment position, and the chromosomes were rearranged. During the second mitotic cycle, only a monopolar spindle was assembled in each blastomere and the cell cleavage was blocked, so that the chromosome set were doubled (Hou et al., 2016). When the induction timing was delayed, the first mitosis was blocked, and abnormal cleavage led to high mortality of embryo (Lin et al., 2015). Meng et al. (2016) gave a protocol of the mitogynogenesis induction in turbot in which the pressure shock of the eggs activated by UV irradiated heterologous sperm initiated at 85–90 mpf with 75 MPa for 6 min. In our induction experiments, we used a different method to determine the initiate time of pressure shock. It was found that the early cleavage of fertilized eggs was affected by many effects, such as water temperature, egg quality and genetic background of fish. And it was also affected by air temperature because of the floating features of turbot eggs. The time of the appearance of cleavage furrow varied from 95 - 110 mpf at 15.0 ± 0.2 °C, which makes it hard
decrease of sperm activity after a long-term preservation and was discarded. The development of fertilized eggs was shown in Fig. 8. There was no distinct difference in morphological traits among mitogynogenetic diploid progenies and control diploids. The hatching period also showed no difference, but the fertilization and hatching rates of mitogynogenetic diploid progenies were lower than those in control diploids (Table 3). About 33.3% hatched larvae survived to 50 dph (1000 individuals from 3000 new hatched larvae, Fig. 8 I). 4. Discussion In the present study, a protocol for artificial induction of mitogynogenetic diploid turbot utilizing homologous sperm was developed. In a number of teleost fish, heterologous sperm has been used to induce gynogenesis because it is usually more convenient to obtain and incapable of producing hybrids with the maternal species (Morgan et al., 2006; Váradi et al., 1999). In gynogenesis induction of flatfish, sperm of the red sea bream and sea bass were widely used because they were easily acquired, and the methods for its cryopreservation and genome inactivation have been well established (Liu et al., 2006). The interspecies gynogenesis was believed to be a way to avoid contamination with radiation induced paternal chromosome fragments (Ocalewicz et al., 2018). However, it has been reported that newly arisen gynogenetic descendants may inherit small parts of paternal genetic material in heterologous sperm initiated embryonic development (Mao et al.,
Fig. 8. The development of mitogynogenetic diploid progenies. A, unfertilized eggs; B, 2 cell stage; C, 4 cell stage; D, 8 cell stage; E, blastula stage; F, gastrula stage; G, crystal stage; H, new hatched larvae; I, juveniles at 50 d post hatching. A-G, scale bar = 0.2 mm; H, scale bar = 0.5 mm; I, scale bar = 5 mm. 6
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to set the exactly induction timing. Therefore, we tried to set the time of the appearance of cleavage furrow, which is more close to the induction timing, as the datum to get a more exact timing. Besides, the fertilized eggs with appearance of cleavage furrow varied from 97 - 102 mpf were used in the optimal treatment parameters determination for the stability and accuracy of the results. By the present induction method, the relative hatching rate could reach more than 10%, which is equal to or far higher than other marine fish or other report in turbot (10 ± 6% in sea bass; 3.2% in turbot; 2.9% in olive flounder; 5.5% in red sea bream) (Francescon et al., 2004; Kato et al., 2001; Meng et al., 2016; Michalik et al., 2015; Zhang et al., 2018). In most teleost species, the optimal pressure intensities varied around 70 MPa (Komen and Thorgaard, 2007). In flatfish, the optimal pressure intensities were 60 MPa in olive flounder (Wang et al., 2008), 70 MPa in half-smooth tongue sole Cynoglossus semilaevis (Chen et al., 2012) and 75 MPa in turbot (Meng et al., 2016), respectively. The optimal pressure intensity of 65 MPa determined in this study is in agreement with most teleost species and other flatfish. While, it is lower than that reported in turbot by Meng et al. (2016). Our results showed that slightly more new hatched larvae could be acquired under high pressure intensity, but most of the new hatched larvae died in the first or second days. And the higher survival rate of new hatched larvae was observed under 65 MPa pressure. So, this pressure intensity might be appropriate. Besides, the optimal pressure intensity may also be affected by egg quality and parental origin, and further study is still needed. With regard to the pressure duration, theoretically it should be related to the speed of embryonic development of the species of fish tested, but for reasons of practicality, an arbitrary shock time of 4–8 min has been applied to most of the reported cases (Komen and Thorgaard, 2007). In our study, only a few haploid larvae hatched and died in a short time. The haploid embryos of turbot cannot survive through the larval stage has also been observed repeatedly in related studies (Meng et al., 2016; Piferrer et al., 2004; Xu et al., 2008). The flow cytometric analysis also proved the purity of haploid and mitogynogenesis. Therefore, it confirmed that all the surviving juveniles from the treatment stocks were mitogynogenetic diploid. Compared with control diploids, mitogynogentic diploids exhibit normal morphology but higher mortality in early development. The decrease in the viability of the gynogenesis has been attributed to their expected high level of inbreeding, which can increase the opportunity for the expression of lethal recessive alleles in their genome (Arai, 2001; Kavumpurath and Pandian, 1994). However, compared to control diploid and meiogynognentic diploid, mitogynognentic diploid is supposed to be fully homozygous. It could greatly accelerate the progress of selective breeding. Considering the relative high fertility of fish, the benefits of mitogynogenesis are greater than the disadvantages in fertility reduction. Mitogynogenesis is preferred to meiogynogenesis in the selection of clone lines with better commercial traits, such as growth performance, due to the homologous. In this study, the mitogynogenesis stocks were cultured at natural seawater temperature in 2 m3, 4 m3 tanks and 16 m3 cement tanks indoor, subsequently. The fast growing period appeared from 12 to 18 mph (from June to December), in which the water temperature was relatively high (21 - 12 °C). There was great divergence of growth performance in 2016 stock. Ten fish in 2016 stock showed obviously higher growth rate at 18 mph, which is similar to the growth rate under industrial production conditions (in net cages with higher water temperature). It was shown that the homozygous individuals with fast growing traits could be screened out only by one generation of mitogynogenesis. The mitogynogenesis induction protocol we established will be useful for generating inbred strains. And the growth performance of the second generation is still under tracking to evaluate the applicability of mitogynogenesis. Female turbot grow faster than male ones as early as 8 mph (Imsland et al., 1997b), and similar phenomenon have also been reported in olive flounder (Yamamoto, 1999b), southern flounder Paralichthys lethostigma (Luckenbach et al., 2004) and half-smooth tongue
sole (Chen et al., 2009). Gynogenesis induction technique can be used to establish homogametic XX male or WW female broodstock (with a sex chromosome system of female homogametic “XX/XY” or male homogametic “ZZ/ZW”, respectively). Therefore, it could be used subsequently for the production of all-females at large scale for commercial farming (Devlin and Nagahama, 2002; Hulata, 2001; Komen and Thorgaard, 2007). Gynogenesis could also be a better way to solve the puzzle of the sex determination system in turbot because the rearing temperature to be reported only has a minor effect on the sex differentiation of juveniles (Haffray et al., 2009). It was assumed that the genetic sex determination mechanism in turbot was female heterogametic ZW/ZZ (Haffray et al., 2009; Martinez et al., 2009; Vinas et al., 2012). In our study, more males than females were observed at all the sampling time in the 2 mitogynogenesis stocks. It is less possible for a homogametic female teleost (XX/XY) than homogametic male teleost (ZW/ZZ) whose sex differentiation is not sensitive to environmental temperature to produce a mitogynogenesis with such a high percentage of males. In male homogamety (ZW/ZZ), the sex ratio of gynogenetic diploid stocks was assumed to be 1:1. However, our results showed a higher male rate in mitogynogenetic diploid than control diploid stocks. It may be caused by the higher mortality of WW offspring. As suggested by Komen and Thorgaard (2007), homogeneous recessive alleles lead to high mortality because of the presence of a lethal locus on the W chromosome close to the sex-determining locus. In half-smooth tongue sole, WW super-females were also found only in the embryos, not in fries (Chen et al., 2012). Our results basically supported the male homogametic “ZZ/ZW” sex chromosome system in turbot. Besides, the rising male ratios were found in mitogynogenetic diploid stocks as well as in control diploid stocks. Considering no high proportion of female deaths was observed and no sex reversal in turbot was reported, we speculated that some other factors, such as environment factors and genetic backgrounds, may cause this phenomenon. The effects of environment factors on sex differentiation still need further study before the sex determination mode is confirmed, and the sex ratios in more families should be investigated. The small numbers of progeny produced by mitogynogenesis surviving to maturity usually displayed dramatically reduced fertility and poor egg quality (Jagiello et al., 2018; Komen and Thorgaard, 2007). The possible reasons might be: (1) embryos of mitogynogenetic diploid are theoretically homozygous at every loci, which include dominance of recessive lethal genes leading to a significant reduction in the survival rate (Felip et al., 2001); (2) the timing of diploidization of chromosome set in mitogynogenesis is later than in meiogynogenesis, which might cause the asynchrony of embryonic development during mitogynogenesis induction, and more severe damage to embryogenesis (Chen et al., 2012). In the present study, the fertilization and hatching rates of mitogynogenetic diploid progenies were also lower than those of control diploid. The asynchronism of mature between male and female may be one of the reasons. Relatively small population size could not provide enough mature males with high quality of semen when females were mature. The mitogynogenetic diploid should be inducted in large scale to obtain more mature fish. Though the fertilization and hatching rates were relatively low in mitogynogenetic diploid progenies, the early development and survival rate were not different from control diploid, showing that the mitogynogenesis is applicable for breeding. In conclusion, we developed an optimal protocol for artificial induction of mitogynogenetic diploid turbot utilizing homologous sperm. The growth performance of mitogynogenesis showed great variation. And fish with fast-growing trait were screened out. Eleven individuals in 2016 mitogynogenesis stock were promoted mature. Though the fertilization and hatching rates reduced in mitogynogenetic diploid progenies, the early development was not obviously different with control diploids. The mitogynogenetic turbot could help to establish pure lines with fast-growing or other traits for commercial farming.
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Declaration of Competing interest
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There are no conflicts of interests in this paper. Acknowledgements This study was supported by the National Natural Science Foundation of China (No. 31772834 and 31502156), National Key R & D Program of China (No. 2018YFD0901202), Key Research and Development Plan of Shandong Province (public welfare special, No. 2017GHY15115), and Industrial Promotion in Agricultural Science and Technology Park of Shandong Province (2017YQ004). References Arai, K., 2001. Genetic improvement of aquaculture finfish species by chromosome manipulation techniques in Japan. Aquaculture 197 (1–4), 205–228. Arai, K., 2002. Significance and prospect of chromosome manipuation in aquaculture of salmonids. Fish. Sci. 68, 734–737. Avise, J.C., 2015. Evolutionary perspectives on clonal reproduction in vertebrate animals. P Natl Acad Sci USA 112, 8867–8873. Bertotto, D., Cepollaro, F., Libertini, A., Barbaro, A., Francescon, A., Belvedere, P., Barbaro, J., Colombo, L., 2005. 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Study on artificial induction, growth and gamete quality of mitogynogenetic turbot Scophthalmus maximus
T
Zhihao Wua,b, Lijuan Wanga,b, Qiaowan Wua,b,c, Yunliang Lud, Zongcheng Songe, Jun Lia,b, Feng Youa,b,∗ a
Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China c University of Chinese Academy of Sciences, Beijing 10049, China d Qingdao Agricultural University, Qingdao 266237, China e Shenghang Aquatic Science and Technology Co. Ltd., Weihai 264200, China b
ARTICLE INFO
ABSTRACT
Keywords: Turbot Scophthalmus maximus Artificial mitogynogenesis induction Growth Sex ratio Offspring
Artificial gynogenesis induction is one of the important techniques of chromosome manipulation. The eggs are fertilized with inactivated sperm to prevent the inheritance from the paternal genome, and genome information of the offspring is totally contributed by the maternal genome. Mitogynogenesis, in which diploidization of chromosome set is performed by blocking the cleavage, is theoretically homozygous and has got more attention in breeding. In this study, we performed the mitogynogenetic diploid induction in turbot Scophthalmus maximus, one of the most important maricultural fish in China and Europe, by applying hydrostatic pressure to the eggs activated with UV-irradiated homologous sperm. The optimal inducing conditions were 36,000 erg mm-2 for UVirradiated and pressure of 65 MPa for 6 min at 15 min before the appearance of the cleavage furrow. Two mitogynogenesis stocks were obtained in 2016 and 2017. The results showed that there was no distinct difference in incubation time and morphological traits between mitogynogenetic diploid and control diploid. The survival rates of mitogynogenetic diploid sharply decreased in the first month post hatching (mph). Their total weight (tW) and total length (tL) increased rapidly from 12 to 18 mph. The mitogynogenetic diploids induced in 2016 and 2017 showed very different growth. The mean tW and tL of 10 fast-growing individuals at 18 mph were 2.78 and 1.32 times of the others in 2016 stock, while 4.50 and 1.74 times in 2017 stock, respectively. The higher male ratio and rising trend of male ratio indicated the female heterogametic ZW/ZZ genetic mechanism of sex determination in turbot. Twenty-four adults in 2017 stock survived until 24 mph. While twenty-nine adults in 2016 stock survived until 36 mph, in which 11 adults could be promoted mature. Then the qualities of gametes were evaluated and showed that both the egg and the sperm qualities of mitogynogenetic diploid were lower than those of control diploid. However, the embryo and larvae development showed no difference between mitogynogenetic diploid progeny and control diploid.
1. Introduction Artificial gynogenesis induction is one of the important techniques of chromosome manipulation. The eggs are fertilized with genetically inactivated sperm to prevent the inheritance from the paternal genome, and genome information of the offspring is totally contributed by the maternal genome (Avise, 2015; Mei and Gui, 2015; Xu et al., 2015). It has been widely utilized in selective breeding, sex control and quantitative trait locus mapping (Arai, 2002; Komen and Thorgaard, 2007). In teleost, the meiogynogenesis can be realized by inhibiting the release of
the second polar body of fertilized eggs and diploidizing the chromosome set in gynogenetic embryos. While the mitogynogenesis can be realized by blocking the early cleavage of fertilized eggs and diploidizing the chromosome set. Until now, most studies focused on artificial induction of meiogynogenesis because the survival rates were relatively high. Besides, meiogynogenetic diploids retain high level of heterozygosity as high frequency of crossover happened in the prophase of the first meiosis (Dayani et al., 2011). On the contrary, mitogynognenetic diploids were theoretically homozygous at almost all loci. It has more application potential in rapidly developing clone lines, genetic
∗ Corresponding author. Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. E-mail address: [email protected] (F. You).
https://doi.org/10.1016/j.aquaculture.2019.734585 Received 4 July 2019; Received in revised form 3 October 2019; Accepted 7 October 2019 Available online 08 October 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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mapping, production of mono-sex stocks and elimination of recessive deleterious genes (Bertotto et al., 2005). However, the survival rate of mitogynogenetic fish was low due to the body deformations or recessive alleles (Jagiello et al., 2017; Polonis et al., 2018). Therefore, artificial mitogynogenesis induction techniques have been developed only in about 20 fish species (Komen and Thorgaard, 2007), such as olive flounder Paralichthys olivaceus (Yamamoto, 1999a; Zhang et al., 2018), red sea bream Pagrus major (Kato et al., 2001), sea bass Dicentrarchus labrax (Francescon et al., 2004) and brown trout Salmo trutta (Michalik et al., 2015). Nowadays, hydrostatic pressure treatment is widely used in induction of mitogynogenesis. The moment of shock induction, pressure intensity and pressure duration are the key factors to determine whether the induction succeed. The moment of shock induction has been confirmed to be the most important factor. Different initiate time of shock induction brought extremely significant difference on induction and survival rates (Lin et al., 2015). Though mitogynogenesis induction may produce higher deformity, lower survival rates, reduced fertility and poor egg quality due to the treatment effects, deleterious recessive mutations and asynchronous embryonic development (Fopp-Bayat et al., 2017; Jagiello et al., 2018; Palti et al., 2002), the benefits of mitogynogenesis on selective breeding overwhelm the defects. The mitogynogenesis with homologous usually showed segregation of characters, such as growth, shape and color, and is an ideal method in the selection of clone lines with better growth or other commercial traits. The screening of different growth performance in gynogenesis has been conducted in cyprinid loach Misgurnus anguillicaudatus (Suzuki et al., 1985), honmoroko Gnathopogon caerulescens (Fujioka, 1998) and fancy carp Cyprinus carpio (Taniguchi et al., 1986). However, almost all such studies based on meiogynogenesis, and the utility of mitogynogenesis in breeding was reported only in a few fish, such as olive flounder (Hou et al., 2016). The studies on the growth performance and the fertility of mitogynogenesis, which are the most important factors in breeding, are still lacking. The turbot Scophthalmus maximus is an important mariculture fish in Europe and China. The annual production in China was 49.5 kilo tons, which is about 83% of the world's total output (FAO, 2018), and makes a principal contribution to the production of land-based tank cultured marine finfish in China (Lei and Liu, 2010). Therefore, the utilization of gynogenesis method in turbot breeding is valuable. Until now, some studies focused on the meiogynogenesis in turbot have been reported (Piferrer et al., 2004; Xu et al., 2008), but only one study gave a protocol of the mitogynogenesis induction in turbot, in which the eggs were activated by UV irradiated heterologous sperm (Meng et al., 2016). The study on growth, gonad development and mature performance of the mitogynogenesis in turbot is lacking, which may define its application in turbot aquaculture and breeding. In the present study, we optimized the mitogynogenesis induction conditions in turbot, and two mitogynogenetic turbot stocks were established by using the optimized condition. The early development, growth performance, sex ratio and gametes quality of the fish in the two stocks were assessed. The results will help in the induction of turbot mitogynogenesis and the breeding application.
be tested within one batch of eggs. 2.2. UV irradiation of turbot sperm The inactivation of turbot sperm was as follows. Semen was diluted to 1:10 in Ringer's solution at 0.0–2.0 °C, then the diluted semen was equally divided into chilled glass Petri dishes (13 cm in diameter), and spread 2 mm in depth. The dishes were placed in a UV box with constant shaking during UV irradiation (254 nm). The UV lamps were warmed up 10 min before onset of the irradiation. The UV exposure was set at 36,000 erg mm-2 under cool conditions (Piferrer et al., 2004). The motility of spermatozoa was checked under microscope before and after the UV irradiation by observing within 20 s. The degree of motility and the average survival rates of each sample were categorized following the reported standard at the temperature of 15.0 ± 0.5 °C. (Strussmann et al., 1994). 2.3. Artificial fertilization To perform artificial fertilization, the irradiated (5 mL diluted) or normal (0.5 mL undiluted) sperm was activated with 15–20 mL filtered seawater at 15.0 °C for 30 s, then was added to plastic beakers containing about 5000 eggs each. After gentle agitation for 30 s, 50–100 mL filtered seawater was added. Then, the eggs were rinsed carefully with seawater at 20 min post fertilization (mpf), and the floating eggs were collected. Eggs fertilized with un-inactivated turbot sperm were used as diploid control. The fertilized eggs were incubated under 15.0 ± 0.2 °C. 2.4. Mitogynogenesis induction The appropriate conditions for the production of mitogynogenetic diploid turbot were examined by varying the moment of hydrostatic pressure shock before the appearance of the cleavage furrow, or by altering the pressure or duration of hydrostatic pressure shock. Hydrostatic pressure induction was carried out in a manual hydrostatic pressure chamber. Three experiments were designed as follows: (1) to determine the optimal moment of shock induction with a single treatment of pressure shock at 65 MPa for 6 min at 5, 10, 15, 20 or 25 min before the appearance of the cleavage furrow; (2) to determine the appropriate pressure intensity of shock with a single treatment of pressure shock at 15 min before the appearance of the cleavage furrow for 6 min at 55, 60, 65, 70 or 75 MPa; (3) to determine the appropriate pressure duration of shock with a single treatment of pressure shock at 15 min before the appearance of the cleavage furrow at 65 MPa for 4, 5, 6, 7 or 8 min. The preset pressure was achieved in 15 s, and recovered to normal pressure in 30 s after shock treatment. Temperature was constantly monitored throughout the experiment. After treatment, shocked eggs were acclimated in seawater for incubation under 15.0 ± 0.2 °C. One male and one female parents were used in each experiment. All experiments were replicated five times using egg batches derived from five different females, and the males were randomly collected from the male stocks containing more than 80 individuals. Fertilization and hatching rates were determined by examining ∼ 200 and 100 floating eggs each experimental group, respectively. Survival rates of larvae indicated the rates of successfully induced larvae. Fertilization rate (%) = buoyant eggs at blastula stage/initial buoyant eggs after induction × 100%; Hatching rate (%) = normal larvae at 12 h after hatching/fertilized eggs × 100%; Survival rate (%) = fertilization rate × hatching rate × 100%
2. Materials and methods 2.1. Brood stock management and gamete collection Turbot brood stocks were cultured in the fish farm of Shenghang Aquatic Science and Technology Co. Ltd., Weihai, Shandong, China under controlled conditions (photoperiod 14 h light: 10 h dark; temperature, 14.0 ± 1.0 °C). The semen was collected from mature male by gently pressuring their abdomens and was stored in 5 mL EP tubes on ice until used for fertilization in 30 min. Semen contaminated with water or urine was discarded. The eggs were stripped from each female, and stored in a wet box under dark condition. The eggs were divided into parts, ∼5000 each before fertilization so that all parameters could
2.5. Assessment of ploidy level The ploidy of control and treatment groups was determined by cellular DNA content and haploid syndrome observation. Flow cytometric analysis with a PARTEC cell counter analyzer CCA-II (PARTEC, Germany) was performed to detect average cellular DNA contents of 2
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Fig. 1. The flow cytometric analysis of control diploid (A), haploid (B) and mitogynogenetic diploid (C) larvae in turbot Scophthalmus maximus.
hatched larvae. Thirty hatched larvae were sampled and prepared in each group (Luckenbach et al., 2004; You et al., 2001). Control diploid larvae were used as diploid standard for the calibration of the cytometer. Similar to other fish, the turbot haploids produced in treatment groups were morphologically abnormal with big heads, short tails or almost tailless. The embryos hardly hatched and died before or soon after hatching, and hence were easily identified by observation.
2.8. Statistical analysis Data from control diploid and mitogynogenesis induction groups were analyzed with the SPSS package for Windows (Version 15.0, SPSS, Chicago, IL). Data are shown as mean ± S.D. Distributions were examined for departures from normality by the Kolmogorov-Smirnov test and the homogeneity of variances was verified by the Levene's test. Significant differences were determined using One-way ANOVA tests followed by Duncan's multiple comparison tests at the probability level of 0.05.
2.6. Growth and sex ratio of mitogynogenetic diploids To assess the growth performance of mitogynogenetic turbot, according to the results of induction conditions, two mitogynogenesis stocks were induced in 2016 and 2017, respectively. The control diploids were also conducted. About 200 mL eggs acquired from 3 - 4 mature female turbot were used for the production of mitogynogenetic diploids. The eggs were fertilized with UV irradiated sperms from 3 - 4 mature male turbot, and then shocked under the optimal parameters. Then eggs were incubated in net cages under 15.0 ± 0.2 °C. The hatched larvae were reared in indoor tanks with flow-through sea water using the protocol for turbot developed by Person-Le et al. (1991) at a temperature of 18.0–21.0 °C, and fed with rotifer, artemia and commercial dry feed (salinity 28–30, pH 7.8–8.2, dissolved oxygen > 6 mg/L, water exchange rate 10% for pre-larvae, 100% for post-larvae and > 800% for juveniles and adults). The culture conditions, such as water parameters, density and feed strategy, were the same as control diploids. From 3 months post hatching (mph) to 24 mph, 30 individuals were randomly sampled from the control diploid and mitogynogenesis stocks every 3 or 6 months, respectively. After the fish were euthanized using MS-222 (60 mg/L, Sigma-Aldrich, Spain), the total length (tL) and total weight (tW) of each fish were measured. The sex of each individual was evaluated with the morphology observation of gonad by using the light through the abdomen from 6 mph to 18 mph. Gonads from five fish for each sex at 6 mph were dissected out, and the sex was confirmed with paraffin section (Ma et al., 2018).
3. Results 3.1. Sperm inactivation UV irradiation caused reduction in the survival of turbot sperm. Duration of sperm motility also declined slightly. After activation by sea water, the motility duration of UV irradiated sperm was about 20 min. The eggs could be fertilized by irradiated sperm. All the embryos exhibited haploid syndrome, and hardly hatched. The ploidy was also confirmed by flow cytometric analysis (Fig. 1). The rarely hatched haploid individual died soon after hatching. 3.2. Optimal parameters of pressure treatment The optimal parameters of pressure treatment were evaluated mainly based on the survival rate. The fertilization and hatching rates in control diploid groups ranged 80.0–98.0% and 60.1–89.7%, respectively. The treatment groups, which the fertilization rates lower than 80.0% or the hatching rates lower than 60.0%, were excluded. The time of the appearance of cleavage furrow in fertilized eggs was around 95–110 mpf under 15.0 ± 0.2 °C, but only the fertilized eggs with appearance of cleavage furrow varied from 97 - 102 mpf were used. Five batches of eggs were applied in each experiment. The fertilization rates of haploid and mitogynogenetic diploid groups were slightly lower than those of control diploid groups due to the inactivation treatment of sperm. A peak of survival rate was obtained when the shock started at 15 min before the appearance of the cleavage furrow, which was significantly higher than those obtained from shocks applied at 5 and 25 min before the appearance of the cleavage furrow (P < 0.05, Fig. 2A). Then some other factors such as shock pressure and shock duration were detected. At 65 MPa, pressure shocks of 6 min brought higher survival rates, and therefore gave the higher production of mitogynogenetic diploid (Fig. 2B and C). In terms of mitogynogenetic diploid production, the optimal conditions were 15 min before the appearance of the cleavage furrow under hydrostatic pressure shock at 65 MPa for 6 min with UV-inactivated turbot sperm.
2.7. Quality assessing of mitogynogenetic turbot gametes The mitogynogenetic turbots over 33 mph were promoted maturity by adjusting the light period and water temperature with nutrition (Imsland et al., 1997a). The male and female gametes were collected from mature fish by gently pressuring their abdomens. Semen from 3 males and eggs from 1 female were obtained. The semen was stored in 5 mL EP tubes on ice until required in 10 min. The eggs were stored at wet box under dark condition. The gamete quality was evaluated according to Valdebenito et al. (2013). The artificial fertilization procedure was the same as the control diploid described in section 2.3. The motility, survival rate and survival time of sperms, morphological traits of eggs, and fertilization and hatching rates of fertilized eggs were estimated for three replicates. The male and female gametes collected from control diploid mature fish from the same origin of mitogynogenetic turbot were also used as the control.
3.3. Production and survival of mitogynogenetic diploid Two mitogynogenetic diploid stocks were induced with the optimal protocol of pressure shock, at 15 min before the appearance of the 3
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Fig. 2. Effects of timing, intensity and duration of hydrostatic pressure shock on the relative fertilization rates and relative survival rates of haploid (n) and mitogynogenetic diploid turbot. Eggs activated with UV-irradiated sperm were shocked at 5–25 min (A) before the appearance of the cleavage furrow at 55–75 MPa (B) for 4–8 min (C); n = 5. The column indicates the relative fertilization rate, and the line indicates the relative survival rate. Data were shown as mean ± S.D.; Different letter indicates significant difference (P < 0.05), the same below. Table 1 The relative fertilization and hatching rates of mitogynogenetic diploid stocks. Stock
Fertilization rate (%)
Hatching rate (%)
2016 2017
71.9 93.3
6.5 6.1
cleavage furrow with 65 MPa shock of 6 min. The relative fertilization and hatching rates in the treatment stocks were shown in Table 1. It took about 120 h for the mitogynogenetic diploids to hatch under 15 °C as well as control diploids. There was no distinct difference in morphological traits of embryos among treatment and control stocks (Fig. 3). The hatching rates of treatment stocks were much lower than those of control stocks. All embryos in haploid stocks showed obvious haploid syndrome. Only a few malformed surviving haploids hatched but died quickly, which ensured the purity of mitogynogenetic diploids (Fig. 3). The ploidy of each stock was also tested by flow cytometry, which presented the acquirement of 100% mitogynogenetic diploid. Compared with control diploid, high mortality occurred during the periods of the first feeding (3 days post hatching, dph) and appearance of swim bladder (about 12 dph) in mitogynogenetic diploid. The survival rates of mitogynogenetic diploid sharply decreased in the first month after hatching, and also obviously decreased at the second month after hatching due to the mortality at metamorphosis period. Fish hardly died after 3 mph (Fig. 4).
Fig. 4. The survival rates of 2 mitogynogenetic diploid (2016 and 2017) and 2 control diploid stocks (2016-2n and 2017-2n).
3.4. Growth performance of mitogynogenetic diploid The growth of 2 mitogynogenetic diploid stocks was showed in Fig. 5. The tW and tL increased rapidly from 12 to 18 mph. Although the 2 stocks showed similar growth trend in the tested period, there were very different growth performance. The mean tW of 10 fastgrowing individuals at 18 mph was 2.78 times of the others induced in 2016, while 4.50 times of the 18 mph individuals induced in 2017. The mean tL of the 10 fast-growing individuals at 18 mph was 1.32 times of the others induced in 2016, while 1.74 times of the 18 mph individuals induced in 2017. Finally, twenty-nine and twenty-four fish respectively survived at 36 and 24 mph in 2016 and 2017 stocks. Both the tW and tL
Fig. 3. The development of control diploid (A), haploid (B) and mitogynogenetic diploid (C) turbot. Scale bar = 0.25 mm. 4
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Fig. 5. The total weight (A, B) and total length (C, D) of 2 mitogynogenetic diploid (2016 and 2017) and 2 control diploid stocks (2016-2n and 2017-2n).
in mitogynogenetic diploid stocks were higher than those in control diploid stocks. The larger S.D. demonstrated larger variation of growth within mitogynogenetic diploid stocks (Fig. 5). 3.5. Sex ratio of mitogynogenetic diploid The sex of each individual was evaluated with the morphology observation of turbot gonad by using the light through the abdomen from 6 mph to 18 mph (Fig. 6). The sex ratios of more than 30 randomly selected fish at 6 mph, and all the fish after 9 mph were checked. The < 60% male ratio was only observed at 6 mph in 2016 and 20162n stocks. A rising trend of male ratio was found in all the stocks, and the male ratios after 9 mph significantly deviated from the 1:1 ratio with a χ2-test (Fig. 7).
Fig. 7. The sex ratios of 2 mitogynogenetic diploid (2016 and 2017) and 2 control diploid stocks (2016-2n and 2017-2n).
3.6. Gametes quality of mitogynogenetic diploid
activated sperms kept high activity (Grade III - IV) for 16.2 ± 3.4 min (Table 2). The unfertilized eggs were buoyant, transparent, spherical in shape, and 1 mm in average diameter with a single oil globule. The first filial generation (F1) stocks were acquired. The fertilization and hatching rates of 2 F1 stocks ranged from 50.0 - 59.1% and 38.0–42.6%, respectively. Another F1 stock showed low fertilization rate due to the
The males which semen could be drawn and the females which the eggs could be stripped were considered to be mature. Nine males and 2 females in 2016 stock were promoted mature on 36 mph. The quality of sperms from three matured males and eggs from one matured female were observed and analyzed. The sperm motility of matured males was around Grade III. About 78.3 ± 2.9% sperms were activated. The
Fig. 6. The distinguishment of turbot sex using the light through the abdomen. A1-A4, male; B1-B4, female; the dotted line indicates the gonad. A1, B1, scale bar = 2 cm; A2, A3, B2, B3, scale bar = 0.5 cm; A4, B4, scale bar = 20 μm. 5
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Table 2 The sperm motility, survival rate and survival time in mitogynogenetic diploid stocks. Replicates
Motility
Survival rate (%)
Survival time (min)
1 2 3 Mean
II-III III-IV III-IV -
80.0 75.0 80.0 78.3 ± 2.9
15.0 13.5 20.0 16.2 ± 3.4
Table 3 The fertilization and hatching rates of mitogynogenetic diploid progenies. Replicates
Fertilization rate (%)
Hatching rate (%)
1 2
59.1 50.0
42.6 38.0
2019), a phenomenon being called leaky gynogenesis according to Janko et al. (2007). Therefore, to avoid unknown effects of heterologous sperm on the offspring, the homologous sperm was utilized in this study. Under the optimized sperm inactivation conditions, 100% gynogenetic haploid could be acquired. The optimal mitogynogenesis induction protocol has also been established in our study. Among the three parameters (timing, intensity and duration) of mitogynogenesis induction with pressure shock treatment, the timing is most critical because it is more species specific than the other two parameters and is usually narrowed down to a range of a very few minutes (Francescon et al., 2004; Komen and Thorgaard, 2007). In the appropriate condition, the spindle was disassembled by the treatment and then resembled in its pretreatment position, and the chromosomes were rearranged. During the second mitotic cycle, only a monopolar spindle was assembled in each blastomere and the cell cleavage was blocked, so that the chromosome set were doubled (Hou et al., 2016). When the induction timing was delayed, the first mitosis was blocked, and abnormal cleavage led to high mortality of embryo (Lin et al., 2015). Meng et al. (2016) gave a protocol of the mitogynogenesis induction in turbot in which the pressure shock of the eggs activated by UV irradiated heterologous sperm initiated at 85–90 mpf with 75 MPa for 6 min. In our induction experiments, we used a different method to determine the initiate time of pressure shock. It was found that the early cleavage of fertilized eggs was affected by many effects, such as water temperature, egg quality and genetic background of fish. And it was also affected by air temperature because of the floating features of turbot eggs. The time of the appearance of cleavage furrow varied from 95 - 110 mpf at 15.0 ± 0.2 °C, which makes it hard
decrease of sperm activity after a long-term preservation and was discarded. The development of fertilized eggs was shown in Fig. 8. There was no distinct difference in morphological traits among mitogynogenetic diploid progenies and control diploids. The hatching period also showed no difference, but the fertilization and hatching rates of mitogynogenetic diploid progenies were lower than those in control diploids (Table 3). About 33.3% hatched larvae survived to 50 dph (1000 individuals from 3000 new hatched larvae, Fig. 8 I). 4. Discussion In the present study, a protocol for artificial induction of mitogynogenetic diploid turbot utilizing homologous sperm was developed. In a number of teleost fish, heterologous sperm has been used to induce gynogenesis because it is usually more convenient to obtain and incapable of producing hybrids with the maternal species (Morgan et al., 2006; Váradi et al., 1999). In gynogenesis induction of flatfish, sperm of the red sea bream and sea bass were widely used because they were easily acquired, and the methods for its cryopreservation and genome inactivation have been well established (Liu et al., 2006). The interspecies gynogenesis was believed to be a way to avoid contamination with radiation induced paternal chromosome fragments (Ocalewicz et al., 2018). However, it has been reported that newly arisen gynogenetic descendants may inherit small parts of paternal genetic material in heterologous sperm initiated embryonic development (Mao et al.,
Fig. 8. The development of mitogynogenetic diploid progenies. A, unfertilized eggs; B, 2 cell stage; C, 4 cell stage; D, 8 cell stage; E, blastula stage; F, gastrula stage; G, crystal stage; H, new hatched larvae; I, juveniles at 50 d post hatching. A-G, scale bar = 0.2 mm; H, scale bar = 0.5 mm; I, scale bar = 5 mm. 6
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to set the exactly induction timing. Therefore, we tried to set the time of the appearance of cleavage furrow, which is more close to the induction timing, as the datum to get a more exact timing. Besides, the fertilized eggs with appearance of cleavage furrow varied from 97 - 102 mpf were used in the optimal treatment parameters determination for the stability and accuracy of the results. By the present induction method, the relative hatching rate could reach more than 10%, which is equal to or far higher than other marine fish or other report in turbot (10 ± 6% in sea bass; 3.2% in turbot; 2.9% in olive flounder; 5.5% in red sea bream) (Francescon et al., 2004; Kato et al., 2001; Meng et al., 2016; Michalik et al., 2015; Zhang et al., 2018). In most teleost species, the optimal pressure intensities varied around 70 MPa (Komen and Thorgaard, 2007). In flatfish, the optimal pressure intensities were 60 MPa in olive flounder (Wang et al., 2008), 70 MPa in half-smooth tongue sole Cynoglossus semilaevis (Chen et al., 2012) and 75 MPa in turbot (Meng et al., 2016), respectively. The optimal pressure intensity of 65 MPa determined in this study is in agreement with most teleost species and other flatfish. While, it is lower than that reported in turbot by Meng et al. (2016). Our results showed that slightly more new hatched larvae could be acquired under high pressure intensity, but most of the new hatched larvae died in the first or second days. And the higher survival rate of new hatched larvae was observed under 65 MPa pressure. So, this pressure intensity might be appropriate. Besides, the optimal pressure intensity may also be affected by egg quality and parental origin, and further study is still needed. With regard to the pressure duration, theoretically it should be related to the speed of embryonic development of the species of fish tested, but for reasons of practicality, an arbitrary shock time of 4–8 min has been applied to most of the reported cases (Komen and Thorgaard, 2007). In our study, only a few haploid larvae hatched and died in a short time. The haploid embryos of turbot cannot survive through the larval stage has also been observed repeatedly in related studies (Meng et al., 2016; Piferrer et al., 2004; Xu et al., 2008). The flow cytometric analysis also proved the purity of haploid and mitogynogenesis. Therefore, it confirmed that all the surviving juveniles from the treatment stocks were mitogynogenetic diploid. Compared with control diploids, mitogynogentic diploids exhibit normal morphology but higher mortality in early development. The decrease in the viability of the gynogenesis has been attributed to their expected high level of inbreeding, which can increase the opportunity for the expression of lethal recessive alleles in their genome (Arai, 2001; Kavumpurath and Pandian, 1994). However, compared to control diploid and meiogynognentic diploid, mitogynognentic diploid is supposed to be fully homozygous. It could greatly accelerate the progress of selective breeding. Considering the relative high fertility of fish, the benefits of mitogynogenesis are greater than the disadvantages in fertility reduction. Mitogynogenesis is preferred to meiogynogenesis in the selection of clone lines with better commercial traits, such as growth performance, due to the homologous. In this study, the mitogynogenesis stocks were cultured at natural seawater temperature in 2 m3, 4 m3 tanks and 16 m3 cement tanks indoor, subsequently. The fast growing period appeared from 12 to 18 mph (from June to December), in which the water temperature was relatively high (21 - 12 °C). There was great divergence of growth performance in 2016 stock. Ten fish in 2016 stock showed obviously higher growth rate at 18 mph, which is similar to the growth rate under industrial production conditions (in net cages with higher water temperature). It was shown that the homozygous individuals with fast growing traits could be screened out only by one generation of mitogynogenesis. The mitogynogenesis induction protocol we established will be useful for generating inbred strains. And the growth performance of the second generation is still under tracking to evaluate the applicability of mitogynogenesis. Female turbot grow faster than male ones as early as 8 mph (Imsland et al., 1997b), and similar phenomenon have also been reported in olive flounder (Yamamoto, 1999b), southern flounder Paralichthys lethostigma (Luckenbach et al., 2004) and half-smooth tongue
sole (Chen et al., 2009). Gynogenesis induction technique can be used to establish homogametic XX male or WW female broodstock (with a sex chromosome system of female homogametic “XX/XY” or male homogametic “ZZ/ZW”, respectively). Therefore, it could be used subsequently for the production of all-females at large scale for commercial farming (Devlin and Nagahama, 2002; Hulata, 2001; Komen and Thorgaard, 2007). Gynogenesis could also be a better way to solve the puzzle of the sex determination system in turbot because the rearing temperature to be reported only has a minor effect on the sex differentiation of juveniles (Haffray et al., 2009). It was assumed that the genetic sex determination mechanism in turbot was female heterogametic ZW/ZZ (Haffray et al., 2009; Martinez et al., 2009; Vinas et al., 2012). In our study, more males than females were observed at all the sampling time in the 2 mitogynogenesis stocks. It is less possible for a homogametic female teleost (XX/XY) than homogametic male teleost (ZW/ZZ) whose sex differentiation is not sensitive to environmental temperature to produce a mitogynogenesis with such a high percentage of males. In male homogamety (ZW/ZZ), the sex ratio of gynogenetic diploid stocks was assumed to be 1:1. However, our results showed a higher male rate in mitogynogenetic diploid than control diploid stocks. It may be caused by the higher mortality of WW offspring. As suggested by Komen and Thorgaard (2007), homogeneous recessive alleles lead to high mortality because of the presence of a lethal locus on the W chromosome close to the sex-determining locus. In half-smooth tongue sole, WW super-females were also found only in the embryos, not in fries (Chen et al., 2012). Our results basically supported the male homogametic “ZZ/ZW” sex chromosome system in turbot. Besides, the rising male ratios were found in mitogynogenetic diploid stocks as well as in control diploid stocks. Considering no high proportion of female deaths was observed and no sex reversal in turbot was reported, we speculated that some other factors, such as environment factors and genetic backgrounds, may cause this phenomenon. The effects of environment factors on sex differentiation still need further study before the sex determination mode is confirmed, and the sex ratios in more families should be investigated. The small numbers of progeny produced by mitogynogenesis surviving to maturity usually displayed dramatically reduced fertility and poor egg quality (Jagiello et al., 2018; Komen and Thorgaard, 2007). The possible reasons might be: (1) embryos of mitogynogenetic diploid are theoretically homozygous at every loci, which include dominance of recessive lethal genes leading to a significant reduction in the survival rate (Felip et al., 2001); (2) the timing of diploidization of chromosome set in mitogynogenesis is later than in meiogynogenesis, which might cause the asynchrony of embryonic development during mitogynogenesis induction, and more severe damage to embryogenesis (Chen et al., 2012). In the present study, the fertilization and hatching rates of mitogynogenetic diploid progenies were also lower than those of control diploid. The asynchronism of mature between male and female may be one of the reasons. Relatively small population size could not provide enough mature males with high quality of semen when females were mature. The mitogynogenetic diploid should be inducted in large scale to obtain more mature fish. Though the fertilization and hatching rates were relatively low in mitogynogenetic diploid progenies, the early development and survival rate were not different from control diploid, showing that the mitogynogenesis is applicable for breeding. In conclusion, we developed an optimal protocol for artificial induction of mitogynogenetic diploid turbot utilizing homologous sperm. The growth performance of mitogynogenesis showed great variation. And fish with fast-growing trait were screened out. Eleven individuals in 2016 mitogynogenesis stock were promoted mature. Though the fertilization and hatching rates reduced in mitogynogenetic diploid progenies, the early development was not obviously different with control diploids. The mitogynogenetic turbot could help to establish pure lines with fast-growing or other traits for commercial farming.
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Declaration of Competing interest
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