Introduction
One of the fish species with the greatest potential for cultivation is the rainbow trout, which has significant potential for worldwide export. It is a cold-water fish belonging to the Salmonidae family, with an optimal growing temperature range of 10–15°C (Shah et al., Reference Shah, Balkhi and Asimi2009). The eyed ova stage in rainbow trout is typically achieved between 14 and 21 days of stripping, and hatching occurs after an additional 9–12 days at 10°C (From and Rasmussen, Reference From and Rasmussen1991). The biological traits that make rainbow trout attractive for culture include their ease of breeding and spawning in captivity, the relative robustness of fry at initial feeding, quick development, high value as a food fish, tolerance to a broad range of water temperatures and other environmental factors (Gall and Crandell, Reference Gall and Crandell1992). Rainbow trout are primarily farmed in cemented raceways, where they account for around 90% of total output (Singh et al., Reference Singh, Pandey and Ali2017). After one year of rearing, the fish typically weigh between 55 to 110 grams; after two years, their weight ranges from 265 to 315 grams (Shah et al., Reference Shah, Balkhi and Asimi2009).
The most common type of polyploidy is triploidy, comprising cells with three sets of homologous chromosomes. Triploids are anticipated to be functionally sterile due to their meiotic inhibition of gametogenesis, which leads to complete suppression of functional sterility and oocyte development. Triploid rainbow trout females have poorly developed gonads in appearance, and ovaries without developing oocytes (Thorgaard and Gall, Reference Thorgaard and Gall1979). Production of triploid individuals can be induced in fish by inhibiting the second meiotic division followed by prevention of second polar body extrusion by shocking eggs shortly after fertilisation (shock treatment causes retention of the second polar body (Thorgaard, Reference Thorgaard1983; Thorgaard, Reference Thorgaard1986; Arai, Reference Arai2001). The odd set of chromosomes is supposed to disrupt oogenesis and prevent communication between germ and somatic cells required for oogenesis (Carrasco et al., Reference Carrasco, Doroshov, Penman and Bromage1998; Krisfalusi et al., Reference Krisfalusi, Wheeler, Thorgaard and Cloud2000; Jagiello et al., Reference Jagiełło, Polonis and Ocalewicz2021). Improved growth rate, enhanced body size, higher food conversion efficiency and sterility are just a few of the traits that triploid fishes display that might be advantageous in commercial fish production (Thorgaard, Reference Thorgaard1983; Preston, Reference Preston2014; Piferrer et al., Reference Piferrer, Beaumont, Falguière, Flajšhans and Haffray2009; Aruljothi, Reference Aruljothi2015, Jagiello et al., Reference Jagiełło, Polonis and Ocalewicz2021). Inducing triploidy in fish involves utilising a wide range of physical shocks and chemical treatments to inhibit the second polar body extrusion during the second meiotic division shortly after mixing of freshly stripped eggs and milt (Solar et al., Reference Solar, Donaldson and Hunter1984; Thorgaard, Reference Thorgaard1986; Ihssen et al., Reference Ihssen, McKay, McMillan and Phillips1990; Felip et al., Reference Felip, Zanuy, Carrillo and Piferrer2001; Komen and Thorgaard, Reference Komen and Thorgaard2007; Preston, Reference Preston2014; Bazaz et al., Reference Bazaz, Nafath-ul-Arab, Ahmad, Shah, Oyas, Yousuf, Bhat, Baba, Fatima and Razak2020; Jagiello et al., Reference Jagiełło, Polonis and Ocalewicz2021). Several approaches, including thermal shock, hydrostatic pressure shock and chemical shock, have been used to elicit triploidy in a variety of fish species, including rainbow trout (Solar et al., Reference Solar, Donaldson and Hunter1984), Ctenopharyngodon idella (Cassani and Caton Reference Cassani and Caton1985), Cyprinus carpio (Linhart et al., Reference Linhart, Flajshans and Kvasnieka1991), red tilapia (Pradeep et al., Reference Pradeep, Srijaya, Papini and Anil2012), brown trout (Preston, Reference Preston2014), and Labeo rohita (Aruljothi, Reference Aruljothi2015). These shock treatments cause destabilisation of microtubules thus affecting centrosomes that are needed to form the mitotic spindle (Komen and Thorgaard, Reference Komen and Thorgaard2007). Consequently, cell division of the egg is interrupted, hence inducing the production of a triploid fish (3n) that has two sets of chromosomes of maternal origin (2n) and a set of chromosomes of paternal origin (1n). Basic knowledge of the number, size and shape of chromosomes has been provided by karyological investigations, which is necessary for chromosomal manipulation and for estimating the ploidy level in fish (Pradeep et al., Reference Pradeep, Srijaya, Papini and Anil2012; Karami et al., Reference Karami, Araghi, Syed and Wilson2015; Bazaz et al., Reference Bazaz, Ahmad, Shah and Arab2022a). Solar et al. (Reference Solar, Donaldson and Hunter1984) reported successful triploid production induced in rainbow trout eggs by heat shock at 1 or 40 min after fertilisation. The most efficient temperatures for the induction of triploidy were 26 and 28°C. All the eggs treated with 3OoC, 40 min after fertilisation died before hatching. Lincoln and Scott (Reference Lincoln and Scott1983) applied 27 to 28oC heat shocks for 15 min, O-45 min after fertilisation. A general review of the results reported seems to indicate that moderate temperature shocks, between 26 and 28°C for 10 to 20 min, within 40 min after fertilisation would be the recommended treatment to suppress polar body extrusion in rainbow trout.
The objective of the present study was to test the influence of the combination of temperature treatments on the triploidy rates and the survival rates of eggs in rainbow trout (O. mykiss).
Materials and methods
The present investigations on triploidy induction involved the following steps for meeting the various objectives:
Collection and segregation of Brood-stock
Trout Culture Farm, Laribal Dachigam, Srinagar (J&K Govt.), provided healthy parent stocks of male and female rainbow trout. To prepare them for spawning, 20 male and female brooders of O. mykiss were kept separately in well-aerated tanks with a continual flow of freshwater. Before sperm and egg collection, the parental broodstock was fasted for 48 h (Bozkurt, Reference Bozkurt2006). The total length of male rainbow trout ranged from 30.3cm to 45.1cm with a mean value of 38.7 ± 1.3 cm while as for female rainbow trout, the length ranged from 34.5cm to 47.4cm with a mean value of 38.0 ± 1.3 cm. The observed total weight of male rainbow trout ranged from 623 g to 1065 g with a mean value of 794.6 ± 49.3 g, while the female rainbow trout weighed in the range of 635 g to 1237g with a mean value of 766.3 ± 64.3 g.
Stripping and fertilisation
For the fertilisation procedure, no inducing hormonal injection was administered. The dry stripping method was used as described by Bozkurt (Reference Bozkurt2006). Using mild pressure on the abdomen, eggs and milt were manually stripped and collected in a clean, sterile and dry plastic container. A delicate, clean bird feather was used to mix the eggs and milt for fertilisation.
Triploidy induction
The methods described by Solar et al. (Reference Solar, Donaldson and Hunter1984), Dillon (Reference Dillon1988) and Dogankaya and Beckan (Reference Dogankaya and Bekcan2014) were used to induce triploidy. The rainbow trout eggs were separated into four treatment units and a separate control group, each group being repeated three times and the number of eggs per replicate taken under experimentation is shown in Table 1.
Table 1. Experimental details
Triploidy induction by heat shock treatment
The eggs were maintained in a hot water bath. An infrared thermometer was used to monitor the temperature of the water bath (Kusam-Meco) (Table 1).
Incubation of eggs
Following the heat shock treatment, the eggs were carefully placed in hatching trays with a continual supply of fresh water for incubation. The water temperature was constantly checked during the incubation period, and dead eggs were taken out and counted every day. The water temperature, that was frequently measured, ranged from 8.3°C to 11.2°C. For each replicate of the treatment group and control group, the fertilisation rate was calculated at the stage of the eyed ova. The hatching rate was calculated when the sac fry (alevin) hatched from the eggs. After 16 to 21 days of stripping, rainbow trout eggs attained the eyed ova stage. Hatching was observed after 34 to 38 days of stripping at 9.5oC water temperature. The fertilisation rate was determined as the percentage of eyed eggs after fertilisation. Similarly, the percentages of alevin (yolk sac fry) and swim-up fry (after yolk sac absorption) were used to calculate hatching and survival rates (Bozkurt, Reference Bozkurt2006).
The following formula was used to calculate the rates of fertilisation, hatching and survival in the control and treatment groups Muir and Robert (Reference Muir and Robert1985).
$$Fertilization\,rate = {{number\,of\,fertilized\,eggs} \over {total\,number\,of\,eggs}} \times 100$$
$$Hatching\,rate = {{number\,of\,hatched\,larvae} \over {number\,of\,fertilized\,eggs}} \times 100$$
$$Survival\,rate = {{number\,of\,survived\,larvae} \over {number\,of\,hatched\,larvae}} \times 100$$
Determination of ploidy
Swim-up fry was used to evaluate the ploidy by preparing the chromosomes according to the methodology proposed by Kligerman & Bloom in Reference Kligerman and Bloom1977, Shao et al. in Reference Shao, Wu, Wang, Tian and Chen2010 and Kenanoglu et al. (Reference Kenanoglu, Yilmaz, Ergun and Aki2013). Karyotyping was carried out for ploidy determination. The ploidy level and the standard treatment combination for triploidy induction were evaluated by counting the chromosomes and estimating the percentage of triploids.
Chromosome types were designated using the methods given by Levan et al. (Reference Levan, Fredga and Sandberg1964) and were categorised as metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a) (Levan et al., Reference Levan, Fredga and Sandberg1964).
Chromosome preparation
Chromosomal spreads were prepared from swim-up fry to identify triploid fish. From each replicate of the heat shock treatment and control groups, 10 swim-up fry were selected randomly. The fry were kept swimming in 500 ml glass beakers containing a 0.05% colchicine solution for 3 to 4 h. After colchicine treatment, the gills and fins were dissected and preserved in 0.56% KCl. The tissue was then diced, homogenised and treated with a hypotonic solution for 25 to 35 min using a glass tissue homogeniser. Following hypotonic treatment, the dissociated tissue was prefixed with a few drops of freshly prepared ice-cold Carnoy’s fixative (3:1 methanol: glacial acetic acid solution) and then centrifuged for 10 min at 1300 rpm. The cell pellet was left undisturbed while the supernatant was discarded. The pellet was mixed with 7 ml of freshly prepared ice-cold Carnoy’s fixative and stored at 4°C. This process was repeated 2 to 3 times at 1 to 2 hour intervals. The samples were kept at 4°C until slide preparation.
Preparation of slides
Using a glass pipette, the cell suspension was dropped onto a clean glass slide. The slides were then allowed to dry at room temperature and stained with 8% Giemsa stain for 20 to 25 min at room temperature. After staining, the slides were washed in distilled water, air dried and then placed in storage for 2 to 3 days before being permanently mounted. With the help of coverslips and DPX mountant, slides were then firmly mounted.
Chromosome analysis
Slides were examined at 10X and 100X (oil emulsion) magnifications to confirm the chromosomal counts. A trinocular microscope (Olympus CX-21) equipped with a camera at 100 X was used to capture microscopic images of the chromosomal spreads.
Statistical analysis
Statistical analysis was done using MS-Excel, Paleontological Statistics Software Package (PAST), Suitable statistical tools such as descriptive/ non-parametric tests including the Kruscal Wallis test, were used for data analysis for determining significance level. Data is presented in mean ± (SE). Pearson’s correlation was estimated.
Result
Fertilisation rate
At the eyed ova stage (Figure 1), rainbow trout fertilisation rates were observed. The mean fertilisation rates for the three replicates in each treatment group – T1, T2, T3 and T4 – were estimated at 90.3 ± 0.3%, 89.4 ± 1.0%, 90.2 ± 0.9% and 88.4 ± 1.6%, respectively. The mean fertilisation rate for the control group (TC) was calculated as 93.1 ± 0.4%. The highest fertilisation rate (90.3 ± 0.3%) was observed in group T1, which was heat-shocked at 26°C, 15 min after fertilisation (TAF) for 10 min. In contrast, the lowest fertilisation rate (88.4 ± 1.6%) was observed in group T4, which was heat-shocked at 28°C, 20 min after fertilisation (Table 2). Fertilisation rates were higher in the control group than in the treatment groups. Table 2 shows a significant difference (P < 0.05) between the treatment and control groups.
Figure 1. Eyed ova stage of rainbow trout (O. mykiss).
Table 2. Fertilisation rates of treatment groups, control group and their replicates in rainbow trout (O. mykiss) after application of heat shock
Hatching rates
The hatching rates for rainbow trout were determined at the sac fry (alevin) stage. The hatching rate (mean ± S.E.) was calculated for each replicate of the treatment and control groups. Hatching occurred 34 to 38 days after stripping at a water temperature of 9.5°C. The estimated mean hatching rates for treatment groups T1, T2, T3 and T4 were 79.7 ± 1.0%, 81.8 ± 0.8%, 72.1 ± 1.1% and 80.9 ± 2.2%, respectively, whereas the hatching rate for the control group (TC) was 84.2 ± 0.4% (Table 3). Among the treatment groups, the T2 group had the highest hatching rate of 81.8 ± 0.8%, while group T3, which was heat-shocked at 28°C for 15 min after fertilisation, had the lowest hatching rate of 72.1 ± 1.1%. Significant differences (P < 0.05) in hatching rates were observed between the treatment groups and the control group, as shown in Table 3.
Table 3. Hatching rates of treatment groups, control group and their replicates of rainbow trout (O. mykiss)
Table 4 illustrates the Pearson relationship between fertilisation rate, hatching rate with heat shock temperature intensity (26 and 28oC) and heat shock time after fertilisation (15 and 20 min.) for rainbow trout. It is observed that temperature intensity showed significant negative correlation with fertilisation rate and hatching rate (r = −0.1, P < 0.01; r = −0.4, P < 0.01), respectively. However, fertilisation rates displayed significant negative (r = −0.3, P < 0.01) and hatching rates showed significant positive correlation with heat shock time after fertilisation (r = 0.6, P < 0.01), respectively.
Table 4. Pearson’s correlation between fertilisation rate, hatching rate, temperature and time after fertilisation in rainbow trout
*Correlation is significant at 0.01 level.
Yolk sac absorption rate
Table 5 presents the yolk sac absorption rate (mean ± S.E.) for each replicate of the treatment and control groups. Complete yolk sac absorption in rainbow trout took an additional 12 to 18 days after hatching (Plate 5). The mean yolk sac absorption rates for treatment groups T1, T2, T3 and T4 in rainbow trout were 76.9 ± 1.2%, 83.9 ± 0.5%, 67.5 ± 0.6% and 79.6 ± 0.8%, respectively. The control group (TC) showed a yolk sac absorption rate of 86.2 ± 0.5%. Significant differences (P < 0.05) in yolk sac absorption rates were observed between the treatment groups and the control group, as shown in Table 5. The group heat-shocked at 26°C after 20 min of fertilisation had the highest yolk sac absorption rate (83.9 ± 0.5%). A significant negative correlation (r = −0.5) was observed between the intensity of the heat shock and the survival rates for complete yolk sac absorption. However, a significant positive correlation (r = 0.7) was established between the time after fertilisation and the survival rates for complete yolk sac absorption.
Table 5. Yolk sac absorption rates of treatment groups, control group and their replicates of rainbow trout (O. mykiss) after application of heat shock
Triploidy rate
In this study, the triploidy rate for each replicate of the treatment groups and the control group was calculated. The (mean ± S.E.) triploidy rates for treatment groups T1, T2, T3 and T4 of rainbow trout were estimated at 26.6 ± 3.3%, 43.3 ± 3.3%, 63.3 ± 3.3% and 76.6 ± 3.3%, respectively. However, no triploids were observed in the control group (Table 6). Significant differences (P < 0.05) in triploidy rates were observed between the treatment groups and the control group, as shown in Table 6. The treatment group T4, which was heat-shocked at 28°C after 20 min of fertilisation, had the highest triploidy rates among the treatment groups in terms of temperature intensity and duration of heat shock initiation. However, in the experimental group T2, which was heat-shocked at 26°C following the 20-minute fertilisation procedure, hatching rates and yolk sac absorption rates were estimated to be higher. No fish from the control group in this investigation were found to be triploid. Time after fertilisation (TAF), temperature intensity and other heat shock parameters significantly affected the percentage of triploidy rates. The TAF had a major impact on the percentage of triploids in rainbow trout, and this study found that triploidy rates were lower at 15 min (26.6%) than at 20 min (43.3%) when the fish were heat-shocked at 26°C. Similarly, a 28°C heat shock applied 15 min after fertilisation resulted in lower triploidy rates (63.3%) compared to a 20-min heat shock (76.6%). The study found that heat shock applied 20 min after fertilisation resulted in greater triploidy rates at both heat shock intensities (26°C and 28°C).
Table 6. Triploidy rates of treatment groups, control group and their replicates of rainbow trout (O. mykiss) after application of heat shock
Table 7 depicts the Pearson correlation between the rate of yolk sac absorption, triploidy rate, heat shock temperature and time after fertilisation in rainbow trout. Yolk sac absorption rate and triploidy rates showed significant positive correlation with heat shock time after fertilisation (r = 0.7, p < 0.01; r = 0.3, p < 0.01), respectively. However, temperature intensity formed significant negative correlation with yolk sac absorption rates (r = −0.5, p < 0.01) while as significant positive correlation was observed between temperature intensity and triploidy rates (r = 0.8, p < 0.01).
Table 7. Pearson correlation between yolk sac absorption rate, triploidy rate, temperature intensity and time after fertilisation in rainbow trout
*Correlation is significant at 0.01 level.
Metacentric (M), submetacentric (SM), subtelecentric (ST), telocentric (T).
Discussion
The present study demonstrates that the control group had significantly higher fertilisation rates than the treatment groups in rainbow trout (O. mykiss). These results are consistent with those of Dogankaya and Bekcan (Reference Dogankaya and Bekcan2014), who found that applying heat shock to rainbow trout eggs at 28°C for 10 min at durations of 10, 15 and 20 min after fertilisation resulted in a significantly lower percentage of live eggs at the eyed stage compared to control groups. Similarly, Saber and Pourkazemi (Reference Saber and Pourkazemi2012) observed that fertilisation rates were lower in treatment groups subjected to heat shocks at 38, 40 and 42°C for 1 min, 4 min after fertilisation, while the control group showed relatively higher fertilisation rates. The current study on O. mykiss aligns with these previous findings.
The lower fertilisation rates observed in heat-shocked groups compared to controls may b attributed to the detrimental effects of thermal shocks, which could be a primary cause of the reduced fertilisation rates (Cherfas et al., Reference Cherfas, Gomelsky, Ben-Dom, Peretz and Hulata1994). The present investigation recorded fertilisation rates in rainbow trout ranging from a minimum of 88.4 ± 1.6% in group T4, heat-shocked at 28°C after 20 min of fertilisation, to a maximum of 90.3 ± 0.3% in group T1, heat-shocked at 26°C after 15 min of fertilisation. The lower fertilisation rates reported in previous research compared to the current study might be linked to differences in the duration of heat shock application.
In the current investigation, the control group had significantly (P < 0.05) higher hatching rates than the treated groups of rainbow trout (O. mykiss). These findings are consistent with those of Dillon (Reference Dillon1988), who reported that rainbow trout eggs heat-treated at 28°C exhibited significantly (P < 0.05) lower hatching rates than eggs heat-treated at 26°C. The results of this study on O. mykiss also align with those of Arai and Wilkins (Reference Arai and Wilkins1987), who found that hatching rates were significantly higher in control groups compared to treated groups. Similar findings have been reported by other studies, including Thorgaard et al. (Reference Thorgaard, Jazwin and Steir1981), Solar et al. (Reference Solar, Donaldson and Hunter1984), Dogankaya and Bekcan (Reference Dogankaya and Bekcan2014) and Bazaz (Reference Bazaz2019). The lower hatching rates observed in heat-shocked eggs compared to controls in this study and others might be primarily attributed to the effects of the heat shock itself (Dillon, Reference Dillon1988).
The current study found that rainbow trout (O. mykiss) exposed to thermal shock treatments had significantly lower survival rates than the control group at the stages of fertilisation, hatching and full yolk sac absorption. These findings are consistent with those of Solar et al. (Reference Solar, Donaldson and Hunter1984), who reported that heat-treated rainbow trout eggs resulted in significantly (P < 0.05) lower survival to feeding compared to controls. Similar results were observed by Dillon (Reference Dillon1988), who found that heat-shocked rainbow trout groups had lower survival rates to feeding than control groups. Kizak et al. (Reference Kızak, Guner, Turel and Kayım2013) also reported that triploid brown trout had lower survival rates than diploid brown trout. Likewise, Dogankaya and Bekcan (Reference Dogankaya and Bekcan2014) found that the survival rates of rainbow trout feeding larvae were considerably lower in the heat-shocked group than in the control group.
The reduced survival rates in the heat-shocked groups may be related to the intensity level of the shock treatment or the retention of the second polar body during triploidy induction, as speculated by Solar et al. (Reference Solar, Donaldson and Hunter1984). Additionally, Babaheydari et al. (Reference Babaheydari, Keyvanshokooha, Dorafshanb and Johari2016) observed changes in the proteome of rainbow trout fertilised eggs, including a reduced abundance of vitellogenin in heat-shock-treated eggs, which may be linked to reduced early survival rates in the heat shock treatment. The experiment was designed to observe the effect of heat shock treatment for inducing triploidy on the proteome changes in fertilised eggs of rainbow trout (O. mykiss).
Reduced abundance of vitellogenin and egg quality in fish can occur, as reported by King et al. (Reference King, Pankhurst, Watts and Pankhurst2003), who investigated the effect of increased temperatures on vitellogenesis and egg quality in female Atlantic salmon. They found that fish kept at 22°C during vitellogenesis had a general reduction in plasma vitellogenin levels compared to fish kept at 14°C and 18°C. In addition, the authors observed an increase in eggshell damage frequency, a decline in maternal investment and a decrease in egg survival. Valdebenito et al. (Reference Valdebenito, Figueroa, Valdebenito and Paiva2021) also found that elevated or fluctuating temperatures during the incubation of eggs caused chorion alterations in the eyed ova stage of salmonid eggs, leading to the most common alteration in farmed salmonid species, known as soft chorion (or soft egg or soft shell disease).
As a result, the reduced vitellogenin levels observed by Babaheydari et al. (Reference Babaheydari, Keyvanshokooha, Dorafshanb and Johari2016) might be associated with increased vitellogenin diffusion due to chorion damage in heat-treated eggs. Therefore, the observed decline in early survival rates during incubation due to heat shock may possibly be the result of vitellogenin diffusion through damaged chorion. The current study supports the observations made by King et al. (Reference King, Pankhurst, Watts and Pankhurst2003) and Babaheydari et al. (Reference Babaheydari, Keyvanshokooha, Dorafshanb and Johari2016), indicating that heat shock has a detrimental effect on the survival of eggs and may be one of the major factors causing eggshell damage with reduced vitellogenin, leading to increased mortality and lower survival rates.
Colchicine, a spindle poison commonly used in fish chromosomal preparation techniques to arrest cells at metaphase, was utilised in this study to confirm triploidy in rainbow trout (Kligerman and Bloom, Reference Kligerman and Bloom1977). Colchicine, a naturally occurring substance in the Colchicum autumnale plant, binds to tubulin, preventing it from polymerising, which disrupts microtubule dynamics and mitosis. When a tubulin dimer binds to colchicine, it causes the polymer to break down and become unstable. After mitotic spindle inhibition, cells or larvae must be treated with a hypotonic solution to enlarge their nuclei and disperse their chromosomes on the slides (Moore and Best, Reference Moore and Best2001). It is crucial to select the appropriate concentration and duration of colchicine treatment, as an insufficient quantity might prevent the target cells from arresting at the metaphase stage, while a high concentration or prolonged exposure could lead to chromosomal condensation (Caperta et al., Reference Caperta, Delgado, Ressurreic, Meister, Jones, Viegas and Houben2006; Bazaz et al., Reference Bazaz, Ahmad, Shah, Bhat, Asimi, Bhat, Arab, Yousuf and Razak2022b).
The current investigation supports the findings of previous studies on salmonid species, which indicated that treatments applied within the first 10 min of the fertilisation process often result in reduced survival rates [Quillet and Gaignon (Reference Quillet and Gaignon1990); Smoker et al. (Reference Smoker, Crandell and Matsuoka1995); Guner et al. (Reference Guner, Peker and Altunok2016)] and in other fish species (Kucharczyk et al., Reference Kucharczyk, Żarski, Targońska, Łuczyński, Szczerbowski, Nowosad, Kujawa and Mamcarz2014). Similar results were observed in this study, showing that applying heat shock 20 min after fertilisation produced more triploids than applying it 15 min after fertilisation. Dillon (Reference Dillon1988) reported that 20 min TAF produced higher rates than 10 min TAF among several heat shock parameters studied on rainbow trout. However, the study also found that heat shocks starting 40 min after fertilisation were ineffective in inducing high triploidy rates, indicating that under ambient conditions, polar body extrusion often occurred before the 40-min mark after fertilisation.
The findings of the present investigation demonstrate that heat shock treatment given to rainbow trout eggs 15 and 20 min after fertilisation at 26°C and 28°C effectively suppressed the second polar body extrusion, resulting in appropriate triploid production rates. These findings are consistent with observations reported in various salmonid species by Solar et al. (Reference Solar, Donaldson and Hunter1984), Johnstone (Reference Johnstone1985), Diaz et al. (Reference Diaz, Iturra, Veloso, Estay and Colihueque1993) and Dogankaya and Bekcan (Reference Dogankaya and Bekcan2014). In this study, rainbow trout (O. mykiss) was found to have 2n = 60 diploid chromosomes and 3n = 91 triploid chromosomes (Figures 2 and 3). The diploid chromosomes of rainbow trout (2n) were classified as 44 metacentric/submetacentric (M/SM), 2 subtelocentric (ST) and 14 telocentric (T) (Figure 4).
Figure 2. Diploid chromosome of rainbow trout (O. mykiss) 2n = 60.
Figure 3. Triploid chromosomes of rainbow trout (O. mykiss) 3n = 91.
Figure 4. Classification of diploid rainbow trout chromosomes 2n (60).
Conclusion
The results of the current investigation showed that the heat shock treatment had a substantial impact on the survival of rainbow trout eggs. In comparison to the treatment groups, the rates of fertilisation, hatching and yolk sac absorption were all significantly (P < 0.05) higher in the control group. It was demonstrated that increasing the heat shock intensity decreased the rates of fertilisation, hatching and yolk sac absorption. The percentage of hatching and yolk sac absorption, however, increased even more as the shock initiation after fertilisation time was extended. Time after fertilisation (TAF), temperature intensity and all heat shock parameters significantly influenced the rate of triploidy. There was a significant (P < 0.05) positive correlation between triploidy rate and temperature intensity and time after fertilisation, demonstrating that as the temperature intensity of the heat shock and the time after fertilisation increased, so did the triploidy rates. The diploid and triploid chromosome numbers in rainbow trout (O. mykiss) were 2n = 60 and 3n = 91, respectively.
The current study concluded that the thermal shock applied 20 min after fertilisation increased triploidy rates at both heat shock intensities (26 and 28°C) and the highest triploidy rate calculated at a heat shock intensity of 28°C applied for 20 min after fertilisation. However, it was observed that a temperature intensity of 26oC applied 20 min after fertilisation was required to achieve the highest hatching rate and yolk sac absorption rates.
Declarations
Authors contributions
All the authors of the present study contributed effectively in the completion of this study.
Competing interests
The authors of the present study declares no competing interest (financial or personal).
Funding
The present study was not funded by any agency/ institution.
Ethical standards
The present study was approved by the advisory committee of Faculty of Fisheries, SKUAST-Kashmir. The authors assert that all procedures contributing to this work comply with the ethical standards of the relevent guidelines of the institute.
