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Comparison of different ploidy detection methods in Oncorhynchus mykiss, the rainbow trout

Comparison of different ploidy detection methods in Oncorhynchus mykiss, the rainbow trout The objective of this study was to determine a simple and reliable ploidy identification protocol for the rainbow trout (RT), Oncorhynchus mykiss, in the field condition. To evaluate the ploidy level and compare different detection protocols, triploid RT and gynogenesis were induced by UV irradiation and/or heat shock. The hatching rate at day 30 was 85.2% and the survival rate at day 90 was 69.4% (fingerling). The sex ratio of female RT was 93.75% in the gynogenesis group, illustrating that the UV irradiation inactivated the sperm DNA. The hatching rate and survival rate were 82.0 and 74.7%, respectively, in the triploid-induced group. The triploid induction rate by heat shock procedure was 73.9%. Cytogenetic protocols for ploidy identification such as chromosome counting, erythrocyte nuclear size comparison, and analysis of nucleolar organizing regions (NORs) by silver staining were compared. Silver nitrate staining showed the greatest success rate (22/23 and 32/32 for the triploid-induced group and gynogenesis group, respectively), followed by erythrocyte nuclear size comparison (16/23 and 19/32 for the triploid-induced group and gynogenesis group, respectively) and, lastly, chromosome preparation (2/23 and 6/32 for the triploid-induced group and gynogenesis group, respectively) with the lowest success rate. Based on our findings, silver staining for RT ploidy identification is speculated to be highly applicable in a wide range of research conditions, due to its cost-effectiveness and simplicity compared to other numerous ploidy detection protocols. Keywords: Rainbow trout, Nucleolar organizing regions, Silver staining, Triploid, Gynogenesis Background (Cal et al. 2006; FAO 2005), meaning that instead of It has been 50 years since the domestication of rainbow sexual maturation, the energy is directed towards the trout (RT) in South Korea, reaching a production of development of flesh quality and somatic growth more than 3000 tons per year (Ministry of Ocean and (Felip et al. 2001; Kizak et al. 2013; Piferrer et al. 2009). Fisheries 2016). However, the lack of a systematic These characteristics have drawn the attention of people control of brood stock, recessive growth due to inbreed- for the preference of triploid fish over diploid. ing, and the increased male ratio are causing the overall Although diploid and triploid fish are morphologically productivity of RT to slump (Hwang 2012). In the global equal throughout their life cycle, they are cytologically differ- aquaculture industry, the induction of numerous ent. Hence, there are many ways, direct or indirect, to iden- artificial triploid fish species is already an important tify the ploidy of a fish (Maxime 2008; Tiwary et al. 2004). subject of study (Felip et al. 1997; Gjedrem et al. 2012; Among those are the measurement of nuclear and cellular Maxime 2008). The usage of triploid fish for industrial size (Alcantar-Vazquez 2016; Thomas and Morrison 1995), purposes has numerous advantages as it contains three electrophoresis of proteins (Liu et al. 1978; Shimizu et al. sets of chromosomes and is genetically sterile. Above all, 1993), nuclear and cell size measurement of erythrocyte these types of fish have reduced gonadal development (Olele and Tiguiri 2013; Pradeep et al. 2011), chromosome counting (Thogaard 1983; Tiwary et al. 1997), DNA content determination with flow cytometry (Alcantar-Vazquez et al. * Correspondence: jhson@noahbio.com 2008; Lamatsch et al. 2000), and staining of nucleoli with Equal contributors Noah Biotech Inc., Cheonan 31035, South Korea silver nitrate (Howell and Black 1980; Porto-Foresti et al. Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. Fisheries and Aquatic Sciences (2017) 20:29 Page 2 of 7 2002). Yet, regardless of type, it is believed that an easy, calculated based on the mass of the container (35.7 l), giving simple, and inexpensive method for ploidy identification is a total of approximately 59,500 eggs. A total of about 47 ml most advantageous and productive. The silver staining of milt was collected from five males and divided into two method for nucleoli identification, nuclear and cell size for the treatment of gynogenesis and triploid production. measurement of erythrocyte, and chromosome counting meet the criterion mentioned above since they are func- Gynogenesis and triploid production tional and have the capacity for a hasty identification of Milt stripped from males was diluted (1:10) with saline solu- ploidy level, whereas most other methods require specific tion and transferred to Petri dishes, 10 cm in diameter, equipments and expensive materials (Carman et al. 1992). forming a thin layer of sperm. The Petri dishes were ex- In this study with RT, we have preferentially focused on sil- posed to UV (Phillips 6 W UV lamp) for 15 min on ice for ver staining over chromosome counting and erythrocyte nu- the inactivation of sperm DNA (Fernandez-Diez et al. 2016). clear size comparison because of two main reasons: first is The eggs were divided into two groups, and each group was randomness. Chromosome preparation is known to be very treated with normal intact milt (triploid-induced group) and random (Deng et al. 2003). There are too many factors to UV-irradiated milt (gynogenesis group) for 2 min and stirred consider such as relative humidity (Spurbeck et al. 1996), with a feather. For every ~ 3000 eggs, 1 ml of milt was used. cell dropping height (Barch et al. 1997; Hlics et al. 1997), After 10 min of fertilization, eggs were exposed to heat and flame vs. air drying method of the slide (Karami et al. shock at 28 °C for 20 min to prevent extrusion of the second 2015). Still, even taking into account all these aspects, polar body. Hatching rate was calculated 30 days post gettinga well-spread metaphaseisoverlytime consuming fertilization, and survival rate was determined as the fish and not always rewarding. This is not an exception with the reached 90 days post fertilization. To further confirm ploidy erythrocyte nuclear size comparison method. Although it is by means of erythrocyte nuclear size, chromosome count- widely used, as mentioned in reports by Felip et al. (2001) ing, and NOR identification, RT fingerlings (n =23, age and Caterina et al. (2014), the nuclear size of red blood cells 3 months old; body weight 1.5–2 g) were randomly selected is not always ~ 1.5 times bigger and it depends on the type and kept alive while being transported to the lab in a 1-gal of anticoagulant used while collecting the blood samples, as dispensing bag connected to an air pump. well as the preservation time of samples and slide prepar- In addition, gonadal tissue slices obtained from the ation conditions. Second is inconsistency in chromosome pool of gynogenesis group fingerlings (n = 32) were set numbers. Due to the Robertsonian translocation in RT onto a slide and gently squashed using a cover glass for chromosome (Inokuchi et al. 1994; Jankun et al. 2007), the sex ratio determination by histological examination change in number is unavoidable. The numbers range from under a microscope. 2n = 56 (Kenanoglu et al. 2013), 2n = 56 to 68 (Oliveira et al. 1995), and 2n = 58 to 63 (Colihueque et al. 2001), making Detection of NORs by silver staining chromosome preparation less reliable. To the contrary, con- Small pieces of fin tissue were obtained without sacrificing sidering there is a direct relationship between the numbers the samples (triploid-induced group), then sheared on a of nucleolar organizing regions (NORs) per chromosome pre-cleaned slide with few drops of 50% acetic acid and pair (Jankun et al. 2007; Phillips et al. 1986) in RT, silver finally let dry in air at room temperature. Samples were staining is a more reliable method of ploidy identification. stained with silver nitrate following the procedures pro- In order to identify polyploidy of the samples (gynogenetic posed by Howell and Black (1980) with a modification to diploid females and presumed triploid RT), three different remove silver residue precipitation. The first solution, solu- ploidy detection methods were compared. Furthermore, tion A (Sol A), was made with 0.5 g of gelatin, 25 ml of hatching rate, survival rates, sex ratio determination, and double-distilled water, and 0.25 ml of formic acid contain- triploid induction rate were also measured. ing formaldehyde (2% final concentration). An aqueous solution, solution B (Sol B), was a mix of 5 g of silver nitrate Methods and 10 ml of double-distilled water. Both Sol A and Sol B Fish were covered with aluminum foil and stored in the dark to RT were randomly selected from Dong Gang Aquaculture avoid photoreaction. As for the staining of the slide, 50 μl located in Pyeongchang. Males (n = 5, length 63.4 ± 2.3 cm; of Sol A and 100 μl of Sol B were dropped on the slide and body weight 3415 ± 576.8 g) and females (n = 19, length the solutions were gently mixed using the side of a pre- 58.6 ± 4.2 cm; body weight 3519 ± 835.7 g) were anesthe- cleaned 3-ml disposable pipette. Next, the slide was placed tized by MS-222 (Tricaine methane sulfonate, 25 mg/l) in a on a hot plate (60 °C) that was covered well to provide as 50-l container. All eggs and milt used in this experiment much darkness as possible for the stain to take place. As were obtained by abdominal massage. Egg quality was evalu- the solution became golden brown, the slide was removed ated by visual inspection. By calculating the average mass of from the hot plate, gently washed under running double- an RT egg (~ 0.6 g), the total number of eggs collected was distilled water, and let dry in air. Kim et al. Fisheries and Aquatic Sciences (2017) 20:29 Page 3 of 7 Chromosome preparation triploid-induced group) and 74.4% (n = 186, gynogenesis Fingerling samples were prepared as described by Kligerman group) at 90 days post fertilization (Table 1). and Bloom (1977) but modified to suit our experiment. To Based on the gonadal tissue examination (Fig. 1), the intercept cell division by interrupting the polymerization of female sex ratio of gynogenesis group was 93.75% microtubules, the fish were transferred into a 2-l glass (30:32), indicating a fairly high induction of female. The beaker and then treated with 0.005% colchicine for 3 h. histological section of female gonadal tissue showed After colchicine treatment, the fish were sacrificed, and fins corrugated structural morphology with signs of imma- and gills were collected and placed in individual 1.5-ml ture oocytes (Fig. 1a). On the other hand, the male testis Eppendorf tubes. Immediately after, samples were treated showed an overall silky surface with immature sperm- with 0.075 M potassium chloride (KCl) hypotonic solution atogonial development (Fig. 1b). for 20 min at room temperature twice. Samples were centri- fuged at 3000 rpm for 2 min, supernatant was removed, and Triploid induction rate Carnoy’s fixative solution (3:1 methanol/acetic acid) was The triploid induction rate measured by silver staining added twice, each lasting 20 min. At the end of the last was 73.9% (17/23, Fig. 2). Throughout our experiments, fixation procedure, samples were stored at 4 °C until assay. we encountered samples with four NORs (Fig. 2e, f), Each sample was placed on a slide with two to three which show similar patterns to the previous results drops of 50% acetic acid. Tissues were gently minced reported by Flajshans et al. (1992) on the existence of into tiny pieces using a 14-gauge needle attached to a four NORs in the course of triploid fish production. 1-ml syringe under a dissecting microscope. After- wards, 7 μl of the minced solution was pipetted and Ploidy identification dropped onto a pre-cleaned slide at a height of The success rate for ploidy identification of each method 30~40 cm and air-dried. The slide was then stained was recorded. Chromosome preparation, erythrocyte with 5% Giemsa for 20 min at room temperature, nuclear size comparison, and silver nitrate staining washed with running double-distilled water, and let dry methods were performed in all samples (gynogenesis in air before observing under the microscope. group and triploid-induced group). The results of each method are shown in Table 2. Chromosome preparation showed a very poor success Erythrocyte nuclear size comparison rate of 6/32 and 2/23 for the gynogenesis group and Due to the difficulty of blood withdrawal from fingerling triploid-induced group, respectively (Table 2). Attaining (3 months old), fish were sacrificed and blood samples a clear image for chromosomal count was very random were aspirated using a 14-gauge needle in a 1-ml syringe (Fig. 3a, b). After many experimental attempts, in which coated with EDTA solution, while preparing the samples we tried our best to maintain a uniform working condi- for chromosome preparation. On a pre-cleaned slide, tion, we were occasionally able to obtain a justifiable 20 μl of blood was placed and smeared using a cover spread of chromosomes. An approximate of 60 chromo- glass. The smeared blood was then stained with 0.22% somes, a characteristic of a diploid cell, was observed Coomassie blue stain (composed of 220 mg Coomassie (Fig. 3b). Incomplete spread of metaphase chromosome, blue in 50 ml methanol, 10 ml acetic acid, and 40 ml disturbing the viewer while performing chromosomal double-distilled water) for 3 min, washed with double- count, is shown in Fig. 3a. distilled water, and let dry in air. Erythrocytes of gynogenesis group and triploid- induced group are shown in Fig. 4. The difference of nu- Microscope and camera equipment clear length of triploid samples from those of diploid All slides were observed using a Zeiss Axiovert 200 was at the major axis as mentioned by Jankun et al. inverted microscope with a magnification of × 600, × 900, (2007). However, the majority of the samples had the and × 1000, and photographs were taken using a Canon tendency to display a minor length difference showing PowerShot G9 digital camera connected to the micro- scope via a Soligor adapter tube. Table 1 Hatching and survival rate of induced gynogenesis and triploid rainbow trout Group Hatching rate Survival rate Results and discussion Days Hatching rate, survival rate, and sex ratio determination 30 90 The average hatching and survival rates were calculated from 250 randomly selected samples of each group. The Gynogenesis group (%) 82.0 (205/250) 74.4 (186/250) hatching rate of triploid-induced group and gynogenesis Triploid-induced group (%) 85.2 (212/250) 69.6 (174/250) group was 85.2% (n = 212) and 82.0% (n = 205), respect- a Days after fertilization ively. The survival rate for each group was 69.6% (n =174, Counted no. of samples/total no. of samples (n = 250) Kim et al. Fisheries and Aquatic Sciences (2017) 20:29 Page 4 of 7 a b Fig. 1 Histological examination of gonadal tissue from rainbow trout. a Section of squeezed female gonadal structure showing signs of primordial oocytes (× 600). b Section of squeezed male gonadal structure showing signs of primordial spermatogonia (× 600) difficulties in ploidy detection. Additionally, although an Phillips and Ihssen (1985) and Phillips et al. (1986) anticoagulant (EDTA) was used to prevent aggregation reported that Oncorhynchus species have only one NOR of erythrocytes, some samples showed signs of coagulation per chromosome pair. Therefore, if the samples from while others displayed signs of hemorrhage (data not the triploid-induced group were triploids, the cells shown). Overall, the success rate for ploidy detection in would be expected to have a maximum of three NORs. erythrocyte nuclear size comparison method was of 19/32 Ploidy detection using silver nitrate was the most and 16/23 for the gynogenesis group and triploid-induced successful (Table 2) compared to the other two methods. group, respectively (Table 2). The results were 32/32 in the gynogenesis group and a b cd e f Fig. 2 Interphase nuclei from rainbow trout stained with silver nitrate. a Triploid O. mykiss sample containing up to three NORs. The excess number of cells but identifiable (× 600). b Ideal triploid sample with a maximum of three NORs (× 600). c, d Diploid O. mykiss sample containing one and two NORs, respectively (× 600). e, f Samples with a maximum of four NORs shown in arrows (× 900 and × 600, respectively) Kim et al. Fisheries and Aquatic Sciences (2017) 20:29 Page 5 of 7 Table 2 Number of successful ploidy detection experiments identification. Although chromosome counting is precise from three different ploidy detection methods and excels to identify different ploidy levels, chromo- Ploidy detection method Group some analysis requires technically sophisticated skill. Furthermore, our study shows that the erythrocyte nu- Gynogenesis Triploid-induced (n = 32) (n = 23) clear size comparison method is, in fact, faster when Chromosome preparation 6/32 2/23 compared to chromosome counting with higher success rate. Yet, we speculated that nuclear size comparison Erythrocyte nuclear size 19/32 16/23 comparison from fish blood cells was, to some degree, subjective and Silver nitrate staining 32/32 22/23 an inaccurate ploidy detection protocol since it depended on numerous factors such as the anticoagulant All ploidy detection methods were tested under identical samples Ploidy-identified sample/total no. of samples used, sample preservation time, and preparation condi- tions (Felip et al. 2001; Caterina et al. 2014). Despite the fact that ploidy identification using silver 22/23 in the triploid-induced group. Moreover, as previ- nitrate in fish specimen is not as widely used as in ously mentioned by Kavalco and Pazza (2004), silver animals, plants, and insects, silver staining for ploidy debris precipitation in the conventional silver nitrate identification is fast and, at the same time, easy and staining procedure is responsible for false positive very reliable since neither special skills nor expensive results, giving difficulties to the viewer when identifying equipments are necessary. There are also several advan- the ploidy of a sample. Nonetheless, our results show tages when identifying the ploidy in RT; for instance, in clearer stains with few or no silver debris reason being place of sacrificing the specimens, samples could be ob- the filtration (0.45 μm) of the staining solution before tained by cutting small pieces of fin from different year- usage. A difference in coloration of stain can be seen in lings and applying the staining method directly in the Fig. 2, which is due to the amount of time exposed to field without the inconvenience of returning to the la- the silver nitrate stain. As reported by Howell and Black boratory. Moreover, this method could be applied in (1980), within 30 s, the stain turns yellow, and within the early embryonic stage and therefore obviate the 2 min, it turns golden brown. Because the time taken for high raising cost and waste of time until being fully the stain to transform into golden brown was not always grown for ploidy identification. According to Phillips et the same, avoiding the stain to become too dark was al. (1986), the majority of these rapidly dividing embry- critical. Through our study, we recommend that the onic cells are composed of their maximum number of optimal staining time should be less than 90 s, because nucleoli, thus making silver staining possible for the longer exposure to the stain would negatively affect the identification of triploids in the early developmental imaging of the sample. stage of fish. Through our study, we have compared and demon- Furthermore, the trial to induce triploid RT from strated three different but easily approachable methods our study [diploid 26.1%; triploid (including those for ploidy detection in RT and, hereinabove, presented with four NORs) 73.9%] is somewhat different from the results (Table 2). the previously reported studies (Hwang 2012). This It can be denoted from our results that all three may be due to the contributed experimental condi- methods have their advantages. However, the most tion’s discrepancies, such as temperature applied to field-applicable, easy, and rapid method of ploidy identi- eggs and the prevention timing of the second polar fication funnels down to silver nitrate staining for NOR body extrusion. ab Fig. 3 Metaphase chromosome from rainbow trout stained with Giemsa. a, b Triploid and diploid chromosomes stained with 5% Giemsa stain under × 900 and × 600, respectively Kim et al. Fisheries and Aquatic Sciences (2017) 20:29 Page 6 of 7 a b Fig. 4 Erythrocyte size comparison. Samples stained with 0.22% Coomassie blue. a Diploid sample (× 1000). b Triploid sample (× 1000) Conclusions Received: 11 August 2017 Accepted: 16 October 2017 According to the data obtained in this study, it is specu- lated that silver staining is a suitable ploidy detection method in RT not only for technically unsophisticated References Alcantar-Vazquez JP. Fisiologia de los peces triploides. 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Comparison of different ploidy detection methods in Oncorhynchus mykiss, the rainbow trout

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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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Abstract

The objective of this study was to determine a simple and reliable ploidy identification protocol for the rainbow trout (RT), Oncorhynchus mykiss, in the field condition. To evaluate the ploidy level and compare different detection protocols, triploid RT and gynogenesis were induced by UV irradiation and/or heat shock. The hatching rate at day 30 was 85.2% and the survival rate at day 90 was 69.4% (fingerling). The sex ratio of female RT was 93.75% in the gynogenesis group, illustrating that the UV irradiation inactivated the sperm DNA. The hatching rate and survival rate were 82.0 and 74.7%, respectively, in the triploid-induced group. The triploid induction rate by heat shock procedure was 73.9%. Cytogenetic protocols for ploidy identification such as chromosome counting, erythrocyte nuclear size comparison, and analysis of nucleolar organizing regions (NORs) by silver staining were compared. Silver nitrate staining showed the greatest success rate (22/23 and 32/32 for the triploid-induced group and gynogenesis group, respectively), followed by erythrocyte nuclear size comparison (16/23 and 19/32 for the triploid-induced group and gynogenesis group, respectively) and, lastly, chromosome preparation (2/23 and 6/32 for the triploid-induced group and gynogenesis group, respectively) with the lowest success rate. Based on our findings, silver staining for RT ploidy identification is speculated to be highly applicable in a wide range of research conditions, due to its cost-effectiveness and simplicity compared to other numerous ploidy detection protocols. Keywords: Rainbow trout, Nucleolar organizing regions, Silver staining, Triploid, Gynogenesis Background (Cal et al. 2006; FAO 2005), meaning that instead of It has been 50 years since the domestication of rainbow sexual maturation, the energy is directed towards the trout (RT) in South Korea, reaching a production of development of flesh quality and somatic growth more than 3000 tons per year (Ministry of Ocean and (Felip et al. 2001; Kizak et al. 2013; Piferrer et al. 2009). Fisheries 2016). However, the lack of a systematic These characteristics have drawn the attention of people control of brood stock, recessive growth due to inbreed- for the preference of triploid fish over diploid. ing, and the increased male ratio are causing the overall Although diploid and triploid fish are morphologically productivity of RT to slump (Hwang 2012). In the global equal throughout their life cycle, they are cytologically differ- aquaculture industry, the induction of numerous ent. Hence, there are many ways, direct or indirect, to iden- artificial triploid fish species is already an important tify the ploidy of a fish (Maxime 2008; Tiwary et al. 2004). subject of study (Felip et al. 1997; Gjedrem et al. 2012; Among those are the measurement of nuclear and cellular Maxime 2008). The usage of triploid fish for industrial size (Alcantar-Vazquez 2016; Thomas and Morrison 1995), purposes has numerous advantages as it contains three electrophoresis of proteins (Liu et al. 1978; Shimizu et al. sets of chromosomes and is genetically sterile. Above all, 1993), nuclear and cell size measurement of erythrocyte these types of fish have reduced gonadal development (Olele and Tiguiri 2013; Pradeep et al. 2011), chromosome counting (Thogaard 1983; Tiwary et al. 1997), DNA content determination with flow cytometry (Alcantar-Vazquez et al. * Correspondence: jhson@noahbio.com 2008; Lamatsch et al. 2000), and staining of nucleoli with Equal contributors Noah Biotech Inc., Cheonan 31035, South Korea silver nitrate (Howell and Black 1980; Porto-Foresti et al. Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. Fisheries and Aquatic Sciences (2017) 20:29 Page 2 of 7 2002). Yet, regardless of type, it is believed that an easy, calculated based on the mass of the container (35.7 l), giving simple, and inexpensive method for ploidy identification is a total of approximately 59,500 eggs. A total of about 47 ml most advantageous and productive. The silver staining of milt was collected from five males and divided into two method for nucleoli identification, nuclear and cell size for the treatment of gynogenesis and triploid production. measurement of erythrocyte, and chromosome counting meet the criterion mentioned above since they are func- Gynogenesis and triploid production tional and have the capacity for a hasty identification of Milt stripped from males was diluted (1:10) with saline solu- ploidy level, whereas most other methods require specific tion and transferred to Petri dishes, 10 cm in diameter, equipments and expensive materials (Carman et al. 1992). forming a thin layer of sperm. The Petri dishes were ex- In this study with RT, we have preferentially focused on sil- posed to UV (Phillips 6 W UV lamp) for 15 min on ice for ver staining over chromosome counting and erythrocyte nu- the inactivation of sperm DNA (Fernandez-Diez et al. 2016). clear size comparison because of two main reasons: first is The eggs were divided into two groups, and each group was randomness. Chromosome preparation is known to be very treated with normal intact milt (triploid-induced group) and random (Deng et al. 2003). There are too many factors to UV-irradiated milt (gynogenesis group) for 2 min and stirred consider such as relative humidity (Spurbeck et al. 1996), with a feather. For every ~ 3000 eggs, 1 ml of milt was used. cell dropping height (Barch et al. 1997; Hlics et al. 1997), After 10 min of fertilization, eggs were exposed to heat and flame vs. air drying method of the slide (Karami et al. shock at 28 °C for 20 min to prevent extrusion of the second 2015). Still, even taking into account all these aspects, polar body. Hatching rate was calculated 30 days post gettinga well-spread metaphaseisoverlytime consuming fertilization, and survival rate was determined as the fish and not always rewarding. This is not an exception with the reached 90 days post fertilization. To further confirm ploidy erythrocyte nuclear size comparison method. Although it is by means of erythrocyte nuclear size, chromosome count- widely used, as mentioned in reports by Felip et al. (2001) ing, and NOR identification, RT fingerlings (n =23, age and Caterina et al. (2014), the nuclear size of red blood cells 3 months old; body weight 1.5–2 g) were randomly selected is not always ~ 1.5 times bigger and it depends on the type and kept alive while being transported to the lab in a 1-gal of anticoagulant used while collecting the blood samples, as dispensing bag connected to an air pump. well as the preservation time of samples and slide prepar- In addition, gonadal tissue slices obtained from the ation conditions. Second is inconsistency in chromosome pool of gynogenesis group fingerlings (n = 32) were set numbers. Due to the Robertsonian translocation in RT onto a slide and gently squashed using a cover glass for chromosome (Inokuchi et al. 1994; Jankun et al. 2007), the sex ratio determination by histological examination change in number is unavoidable. The numbers range from under a microscope. 2n = 56 (Kenanoglu et al. 2013), 2n = 56 to 68 (Oliveira et al. 1995), and 2n = 58 to 63 (Colihueque et al. 2001), making Detection of NORs by silver staining chromosome preparation less reliable. To the contrary, con- Small pieces of fin tissue were obtained without sacrificing sidering there is a direct relationship between the numbers the samples (triploid-induced group), then sheared on a of nucleolar organizing regions (NORs) per chromosome pre-cleaned slide with few drops of 50% acetic acid and pair (Jankun et al. 2007; Phillips et al. 1986) in RT, silver finally let dry in air at room temperature. Samples were staining is a more reliable method of ploidy identification. stained with silver nitrate following the procedures pro- In order to identify polyploidy of the samples (gynogenetic posed by Howell and Black (1980) with a modification to diploid females and presumed triploid RT), three different remove silver residue precipitation. The first solution, solu- ploidy detection methods were compared. Furthermore, tion A (Sol A), was made with 0.5 g of gelatin, 25 ml of hatching rate, survival rates, sex ratio determination, and double-distilled water, and 0.25 ml of formic acid contain- triploid induction rate were also measured. ing formaldehyde (2% final concentration). An aqueous solution, solution B (Sol B), was a mix of 5 g of silver nitrate Methods and 10 ml of double-distilled water. Both Sol A and Sol B Fish were covered with aluminum foil and stored in the dark to RT were randomly selected from Dong Gang Aquaculture avoid photoreaction. As for the staining of the slide, 50 μl located in Pyeongchang. Males (n = 5, length 63.4 ± 2.3 cm; of Sol A and 100 μl of Sol B were dropped on the slide and body weight 3415 ± 576.8 g) and females (n = 19, length the solutions were gently mixed using the side of a pre- 58.6 ± 4.2 cm; body weight 3519 ± 835.7 g) were anesthe- cleaned 3-ml disposable pipette. Next, the slide was placed tized by MS-222 (Tricaine methane sulfonate, 25 mg/l) in a on a hot plate (60 °C) that was covered well to provide as 50-l container. All eggs and milt used in this experiment much darkness as possible for the stain to take place. As were obtained by abdominal massage. Egg quality was evalu- the solution became golden brown, the slide was removed ated by visual inspection. By calculating the average mass of from the hot plate, gently washed under running double- an RT egg (~ 0.6 g), the total number of eggs collected was distilled water, and let dry in air. Kim et al. Fisheries and Aquatic Sciences (2017) 20:29 Page 3 of 7 Chromosome preparation triploid-induced group) and 74.4% (n = 186, gynogenesis Fingerling samples were prepared as described by Kligerman group) at 90 days post fertilization (Table 1). and Bloom (1977) but modified to suit our experiment. To Based on the gonadal tissue examination (Fig. 1), the intercept cell division by interrupting the polymerization of female sex ratio of gynogenesis group was 93.75% microtubules, the fish were transferred into a 2-l glass (30:32), indicating a fairly high induction of female. The beaker and then treated with 0.005% colchicine for 3 h. histological section of female gonadal tissue showed After colchicine treatment, the fish were sacrificed, and fins corrugated structural morphology with signs of imma- and gills were collected and placed in individual 1.5-ml ture oocytes (Fig. 1a). On the other hand, the male testis Eppendorf tubes. Immediately after, samples were treated showed an overall silky surface with immature sperm- with 0.075 M potassium chloride (KCl) hypotonic solution atogonial development (Fig. 1b). for 20 min at room temperature twice. Samples were centri- fuged at 3000 rpm for 2 min, supernatant was removed, and Triploid induction rate Carnoy’s fixative solution (3:1 methanol/acetic acid) was The triploid induction rate measured by silver staining added twice, each lasting 20 min. At the end of the last was 73.9% (17/23, Fig. 2). Throughout our experiments, fixation procedure, samples were stored at 4 °C until assay. we encountered samples with four NORs (Fig. 2e, f), Each sample was placed on a slide with two to three which show similar patterns to the previous results drops of 50% acetic acid. Tissues were gently minced reported by Flajshans et al. (1992) on the existence of into tiny pieces using a 14-gauge needle attached to a four NORs in the course of triploid fish production. 1-ml syringe under a dissecting microscope. After- wards, 7 μl of the minced solution was pipetted and Ploidy identification dropped onto a pre-cleaned slide at a height of The success rate for ploidy identification of each method 30~40 cm and air-dried. The slide was then stained was recorded. Chromosome preparation, erythrocyte with 5% Giemsa for 20 min at room temperature, nuclear size comparison, and silver nitrate staining washed with running double-distilled water, and let dry methods were performed in all samples (gynogenesis in air before observing under the microscope. group and triploid-induced group). The results of each method are shown in Table 2. Chromosome preparation showed a very poor success Erythrocyte nuclear size comparison rate of 6/32 and 2/23 for the gynogenesis group and Due to the difficulty of blood withdrawal from fingerling triploid-induced group, respectively (Table 2). Attaining (3 months old), fish were sacrificed and blood samples a clear image for chromosomal count was very random were aspirated using a 14-gauge needle in a 1-ml syringe (Fig. 3a, b). After many experimental attempts, in which coated with EDTA solution, while preparing the samples we tried our best to maintain a uniform working condi- for chromosome preparation. On a pre-cleaned slide, tion, we were occasionally able to obtain a justifiable 20 μl of blood was placed and smeared using a cover spread of chromosomes. An approximate of 60 chromo- glass. The smeared blood was then stained with 0.22% somes, a characteristic of a diploid cell, was observed Coomassie blue stain (composed of 220 mg Coomassie (Fig. 3b). Incomplete spread of metaphase chromosome, blue in 50 ml methanol, 10 ml acetic acid, and 40 ml disturbing the viewer while performing chromosomal double-distilled water) for 3 min, washed with double- count, is shown in Fig. 3a. distilled water, and let dry in air. Erythrocytes of gynogenesis group and triploid- induced group are shown in Fig. 4. The difference of nu- Microscope and camera equipment clear length of triploid samples from those of diploid All slides were observed using a Zeiss Axiovert 200 was at the major axis as mentioned by Jankun et al. inverted microscope with a magnification of × 600, × 900, (2007). However, the majority of the samples had the and × 1000, and photographs were taken using a Canon tendency to display a minor length difference showing PowerShot G9 digital camera connected to the micro- scope via a Soligor adapter tube. Table 1 Hatching and survival rate of induced gynogenesis and triploid rainbow trout Group Hatching rate Survival rate Results and discussion Days Hatching rate, survival rate, and sex ratio determination 30 90 The average hatching and survival rates were calculated from 250 randomly selected samples of each group. The Gynogenesis group (%) 82.0 (205/250) 74.4 (186/250) hatching rate of triploid-induced group and gynogenesis Triploid-induced group (%) 85.2 (212/250) 69.6 (174/250) group was 85.2% (n = 212) and 82.0% (n = 205), respect- a Days after fertilization ively. The survival rate for each group was 69.6% (n =174, Counted no. of samples/total no. of samples (n = 250) Kim et al. Fisheries and Aquatic Sciences (2017) 20:29 Page 4 of 7 a b Fig. 1 Histological examination of gonadal tissue from rainbow trout. a Section of squeezed female gonadal structure showing signs of primordial oocytes (× 600). b Section of squeezed male gonadal structure showing signs of primordial spermatogonia (× 600) difficulties in ploidy detection. Additionally, although an Phillips and Ihssen (1985) and Phillips et al. (1986) anticoagulant (EDTA) was used to prevent aggregation reported that Oncorhynchus species have only one NOR of erythrocytes, some samples showed signs of coagulation per chromosome pair. Therefore, if the samples from while others displayed signs of hemorrhage (data not the triploid-induced group were triploids, the cells shown). Overall, the success rate for ploidy detection in would be expected to have a maximum of three NORs. erythrocyte nuclear size comparison method was of 19/32 Ploidy detection using silver nitrate was the most and 16/23 for the gynogenesis group and triploid-induced successful (Table 2) compared to the other two methods. group, respectively (Table 2). The results were 32/32 in the gynogenesis group and a b cd e f Fig. 2 Interphase nuclei from rainbow trout stained with silver nitrate. a Triploid O. mykiss sample containing up to three NORs. The excess number of cells but identifiable (× 600). b Ideal triploid sample with a maximum of three NORs (× 600). c, d Diploid O. mykiss sample containing one and two NORs, respectively (× 600). e, f Samples with a maximum of four NORs shown in arrows (× 900 and × 600, respectively) Kim et al. Fisheries and Aquatic Sciences (2017) 20:29 Page 5 of 7 Table 2 Number of successful ploidy detection experiments identification. Although chromosome counting is precise from three different ploidy detection methods and excels to identify different ploidy levels, chromo- Ploidy detection method Group some analysis requires technically sophisticated skill. Furthermore, our study shows that the erythrocyte nu- Gynogenesis Triploid-induced (n = 32) (n = 23) clear size comparison method is, in fact, faster when Chromosome preparation 6/32 2/23 compared to chromosome counting with higher success rate. Yet, we speculated that nuclear size comparison Erythrocyte nuclear size 19/32 16/23 comparison from fish blood cells was, to some degree, subjective and Silver nitrate staining 32/32 22/23 an inaccurate ploidy detection protocol since it depended on numerous factors such as the anticoagulant All ploidy detection methods were tested under identical samples Ploidy-identified sample/total no. of samples used, sample preservation time, and preparation condi- tions (Felip et al. 2001; Caterina et al. 2014). Despite the fact that ploidy identification using silver 22/23 in the triploid-induced group. Moreover, as previ- nitrate in fish specimen is not as widely used as in ously mentioned by Kavalco and Pazza (2004), silver animals, plants, and insects, silver staining for ploidy debris precipitation in the conventional silver nitrate identification is fast and, at the same time, easy and staining procedure is responsible for false positive very reliable since neither special skills nor expensive results, giving difficulties to the viewer when identifying equipments are necessary. There are also several advan- the ploidy of a sample. Nonetheless, our results show tages when identifying the ploidy in RT; for instance, in clearer stains with few or no silver debris reason being place of sacrificing the specimens, samples could be ob- the filtration (0.45 μm) of the staining solution before tained by cutting small pieces of fin from different year- usage. A difference in coloration of stain can be seen in lings and applying the staining method directly in the Fig. 2, which is due to the amount of time exposed to field without the inconvenience of returning to the la- the silver nitrate stain. As reported by Howell and Black boratory. Moreover, this method could be applied in (1980), within 30 s, the stain turns yellow, and within the early embryonic stage and therefore obviate the 2 min, it turns golden brown. Because the time taken for high raising cost and waste of time until being fully the stain to transform into golden brown was not always grown for ploidy identification. According to Phillips et the same, avoiding the stain to become too dark was al. (1986), the majority of these rapidly dividing embry- critical. Through our study, we recommend that the onic cells are composed of their maximum number of optimal staining time should be less than 90 s, because nucleoli, thus making silver staining possible for the longer exposure to the stain would negatively affect the identification of triploids in the early developmental imaging of the sample. stage of fish. Through our study, we have compared and demon- Furthermore, the trial to induce triploid RT from strated three different but easily approachable methods our study [diploid 26.1%; triploid (including those for ploidy detection in RT and, hereinabove, presented with four NORs) 73.9%] is somewhat different from the results (Table 2). the previously reported studies (Hwang 2012). This It can be denoted from our results that all three may be due to the contributed experimental condi- methods have their advantages. However, the most tion’s discrepancies, such as temperature applied to field-applicable, easy, and rapid method of ploidy identi- eggs and the prevention timing of the second polar fication funnels down to silver nitrate staining for NOR body extrusion. ab Fig. 3 Metaphase chromosome from rainbow trout stained with Giemsa. a, b Triploid and diploid chromosomes stained with 5% Giemsa stain under × 900 and × 600, respectively Kim et al. Fisheries and Aquatic Sciences (2017) 20:29 Page 6 of 7 a b Fig. 4 Erythrocyte size comparison. Samples stained with 0.22% Coomassie blue. a Diploid sample (× 1000). b Triploid sample (× 1000) Conclusions Received: 11 August 2017 Accepted: 16 October 2017 According to the data obtained in this study, it is specu- lated that silver staining is a suitable ploidy detection method in RT not only for technically unsophisticated References Alcantar-Vazquez JP. Fisiologia de los peces triploides. 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Fisheries and Aquatic SciencesSpringer Journals

Published: Nov 8, 2017

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