Introduction
Androgenesis is a reproductive process of producing offspring composed of only paternal genetic materials, therefore resulting in all-male progenies (Zhou et al., Reference Zhou, Wang, Liu, Chen, Sun, Zheng, Mahboob, Xu, Jiang, Zhuang and Jin2019 ). It has been applied to obtain progeny of high inbred lines/clones and to investigate the interrelationship of the nucleus and cytoplasm (Hussain, Reference Hussain1998). This reproductive strategy has also received special attention due to its applicability for restoration of rare and disappearing species using only the genetic material of the male (Birstein et al., Reference Birstein, Bemis, Waldman and Birstein1997). The application for selective breeding programmes, gene mapping and genome sequencing procedures, as well as the production of monosex all-male fish groups, has been exploited extensively in commercially cultured fish species (Komen and Thorgaard, Reference Komen and Thorgaard2007). In particular, this method of monosex production has found ‘pride of place’ among other technologies popularly considered for sex control in fish, such as hormone administration, hybridization and triploidy induction (Piferrer, Reference Piferrer2001; Beardmore et al., Reference Beardmore, Mair and Lewis2001).
Although sex reversal by hormonal administration of steroids (e.g. 17α-methyltestosterone or testosterone) only turns genetic female progeny into functional phenotypic males, many countries have raised objections to their commercial use in food fish due to the fear that it could affect human health (Hunter and Donaldson, Reference Hunter, Donaldson, Hoar, Randall and Donaldson1983; Tave, Reference Tave1992; Okomoda et al., Reference Okomoda, Pradeep, Oladimeji, Abol-Munafi, Alabi, Ikhwanuddin, Martins, Umaru and Hassan2020a). Unlike androgenesis, the versatility of the use of hybridization and triploidy induction processes for monosex production has not been convincingly demonstrated in many popular aquaculture fish species. Therefore, induction of androgenesis can serve as an alternative and reliable monosex production technique for commercial application in aquaculture (Olufeagba and Moses, Reference Olufeagba and Moses2011). Research techniques for the production of artificial isogenic lines through uniparental inheritance were first established by Purdom (Reference Purdom1969) in plaice Pleuronectes platessa and Japanese flounder Paralichthys olivaceus. These techniques have since been applied to several fish species, many of which were of aquaculture importance (Komen and Thorgaard, Reference Komen and Thorgaard2007; Hou et al., Reference Hou, Saito, Fujimoto, Yamaha and Arai2014, Reference Hou, Wang, Zhang, Sun, Liu and Wang2016; Balashov et al., Reference Balashov, Vinogradov, Kovalev, Barmintseva, Recoubratsky and Grunina2017; Iegorova et al., Reference Iegorova, Psenicka, Lebeda, Rodina and Saito2018; Polonis et al., Reference Polonis, Jagiełło, Dobosz, Rożyński, Kondraciuk, Gurgul, Szmatoła and Ocalewicz2019).
Several methods have been used in the artificial production of androgenetic organisms. A less popular method of induction involves cold-shock inhibition of the extrusion of the second polar body, resulting in a later extrusion of the same, together with the egg pronucleus (Morishima et al., Reference Morishima, Fujimoto, Sato, Kawae, Zhao, Yamaha and Arai2011; Hou et al., Reference Hou, Saito, Fujimoto, Yamaha and Arai2014, Reference Hou, Wang, Zhang, Sun, Liu and Wang2016; Iegorova et al., Reference Iegorova, Psenicka, Lebeda, Rodina and Saito2018). The most common method of androgenetic induction involves two main steps, i.e. irradiation of the egg nucleus and doubling of paternal chromosomes (Balashov et al., Reference Balashov, Vinogradov, Kovalev, Barmintseva, Recoubratsky and Grunina2017). Gamma, ultraviolet (UV) and X-rays are used to destroy the genetic material of the egg and, when activated by a sperm, it results in the development of a haploid androgenetic embryo (Polonis et al., Reference Polonis, Jagiełło, Dobosz, Rożyński, Kondraciuk, Gurgul, Szmatoła and Ocalewicz2019). Subsequent exposure of this haploid egg to shock at about the time of the prophase of the first mitotic division results in the prevention of cell cleavage, hence the duplication of the paternal chromosomes to restore the diploid status of the fish (Bongers et al., Reference Bongers, in ’t Veld, Abo-Hashema, Bremmer, Eding, Komen and Richter1994; Reference Bongers, Zandieh-Doulabi, Richter and Komen1999; Ocalewicz et al., Reference Ocalewicz, Dobosz, Kuzminski, Nowosad and Goryczko2010; Nowosad et al., Reference Nowosad, Kucharczyk, Liszewski, Targońska and Kujawa2015). Studies with rainbow trout Oncorhynchus mykiss (Thorgaard et al., Reference Thorgaard, Scheerer, Hershberger and Myers1990) and loach Misgurnus anguillicaudatus (Arai et al., Reference Arai, Ikeno and Suzuki1995) have, however, produced androgenetic diploids through an alternative method of fertilizing irradiated eggs with sperm of tetraploid males (4n). This, therefore, removes the need for a shock protocol, necessary in restoring the diploid status of the developing embryo when activation is carried out with sperm from diploid males.
The African catfish Clarias gariepinus is a freshwater fish species of aquaculture importance as it is popularly cultured in many nations and contributes significantly to the local economies (Volckaert et al., Reference Volckaert, Galbusera, Hellemans, Van den Haute, Vanstaeri and Ollevier1994). Although the fish has no problem with precarious early reproduction, the male progeny of the African catfish are known to grow faster and reach a larger final size than females (Enuekwe and Okonji, Reference Enuekwe and Okonji2019). Aside from the better performance characteristics under captive conditions, such as better feed conversion and protein efficiency ratios (Turan and Akyurt, Reference Turan and Akyurt2005), many consumers also seem to prefer male African catfish to their female counterparts. This is because of the high fillet yield from males, resulting from a substantially low gut and gonad weight (≤6% of total weight) at the market size compared with females (about 20% of the total weight) of the same size (Penman, Reference Penman2016). Hence, the production of all-male progeny of C. gariepinus through androgenesis would be very advantageous to catfish farmers. Similarly, the African catfish is used as a good animal model in many developmental, environmental and toxicological studies (Sule and Adikwu, Reference Sule and Adikwu2004; Hassan et al., Reference Hassan, Okomoda and Nurhayati2018a).
Similar to other chromosome manipulation protocols (e.g. Pandian and Koteeswaran, Reference Pandian and Koteeswaran1998; Felip et al., Reference Felip, Zanuy, Carillo, Martinez, Ramos and Piferrer1997; Piferrer et al., Reference Piferrer, Beaumont, Falguiere, Flajshans, Haffray and Colombo2009; Okomoda et al., Reference Okomoda, Aminath, Oladimeji, Abol-Munafi, Alabi, Ikhwanuddin, Umaru, Hassan, Martins and Shahreza2020b), the processes of androgenesis is complicated by many variables that need to be optimized, among which is the duration, intensity and distance of irradiation. An earlier study by Bongers et al. (Reference Bongers, in ’t Veld, Abo-Hashema, Bremmer, Eding, Komen and Richter1994) had optimized UV doses and duration of irradiation for African catfish eggs at the same irradiation distance. In this study, therefore, we attempted to optimize the irradiation distance and time of exposure using the same irradiation intensity for artificial induction of androgenesis in African catfish C. gariepinus.
Materials and methods
Broodstock conditioning and collection of gamete
Thirty mature African catfish weighing between 1–2 kg (sex ratio 1:1) were obtained from Setiu fish farm in Terengganu, Malaysia and were acclimatized for 2 weeks in two 8000-litre tanks filled halfway with fresh water; male and female broodstock were separated in different tanks to avoid predation along sex lines. Water quality was maintained within the optimum range (i.e. temperature 26–27°C, dissolved oxygen (DO) >5 mg/L and pH 5–7) and the fish were fed a commercial diet [45% crude protein (CP)]. The approval for the experimental protocols used in the study was given by the Universiti Malaysia Terengganu committee on research. Hence, all methods applied that pertained to animal care and use complied with specified international, national and institutional guidelines.
Induced breeding of the catfish was performed following the methods of Hassan et al. (Reference Hassan, Okomoda and Nurhayati2018a) and Okomoda et al. (Reference Okomoda, Pradeep, Oladimeji, Abol-Munafi, Alabi, Ikhwanuddin, Martins, Umaru and Hassan2020a). Briefly, six females were injected with synthetic hormone (Ovaprim® at 0.5 ml kg-1) and maintained for a latency period of 9 h before attempting to strip the eggs. All female eggs were collected in a clean bowl by gently applying pressure along the abdomen of the fish. The pooled eggs were homogenized together by gentle mixing using a chicken feather. The male broodstocks (six in number) were then sacrificed by dissecting their lower abdomen to remove the testes from their body cavity. The testes were all cut into small pieces using a pair of scissors and the milt squeezed out using a muslin cloth into three 15 ml conical-bottomed centrifuge test tubes. Samples were then stored on ice through the process of induction.
Ultraviolet irradiation, egg fertilization and shock induction
UV irradiation was carried out in a dark room to avoid genetic photoreactivation (Christopher et al., Reference Christopher, Murugesan and Sukumaran2012). Four glass UV irradiation chambers were constructed each with a UV light bulb (Model UVGL-15 Mineralight® multi-band light) emitting 254/366 nm short-wave radiation fixed at the four different distances designed for this current study. Each irradiation chamber was also equipped with a stopwatch to accurately determine the duration of egg exposure. Approximately 1 g of the homogenized eggs containing approximately 600 eggs were distributed into 60 Petri dish for optimization of UV distance (i.e. 5, 10, 20 and 30 cm) at different durations (1, 2, 3, 4 and 5 min) as shown in Table 1. This means that 20 combinations of irradiation distance and duration (i.e. 20 treatment groups) were observed in triplicate for this study. Following the recommendations of Bonger et al. (Reference Bongers, in ’t Veld, Abo-Hashema, Bremmer, Eding, Komen and Richter1994), 1 ml of Ringer’s solution was introduced into all the Petri dishes containing the eggs to maintain a single layer for an effective irradiation process. The use of Ringer’s solution has been demonstrated to be effective in dissociating the adhesive nature of the fish eggs without activating them (Lin and Dabrowski, Reference Lin and Dabrowski1998). Thereafter, the irradiated egg samples were mixed with 0.5 ml of the stored sperm and activated with water for the purpose of fertilization. Each replicate was fertilized uniquely by sperm from only one of the 15 ml centrifuge test tubes earlier stored to ensure that all treatments were fertilized from a similar sperm pool, hence reducing bias in the study.
Treatment combinations of irradiation distances and duration used for the study
Note: all treatments groups were cold shocked at 5°C for 5 min just moments before the cell cleavage.
Breeding performance of eggs of Clarias gariepinus irradiated at various distances for various times (UV wave of 254/366 nm). Numbers are means ± standard errors
* Unable to assess diploid percentage either due to complete mortality of progenies or insufficient progenies (<10) survival at characterization. Mean in the same column with different superscripts differ significantly (P ≤ 0.05).
Next, the fertilized eggs of the treatment groups were cold shocked at 5°C for 5 min just moments before cell cleavage (about 30–32 min post-fertilization). To briefly describe the cold-shock treatment: eggs were placed in a polyvinyl chloride (PVC) cylinder (measuring 12 cm diameter × 18 cm high) fitted with a fine mesh net (<0.5 mm openings) attached to the bottom of the PVC and immersed in three already prepared tanks (for each replicated group per treatment) measuring 1 × 1 × 0.5 m3 and containing ice cubes/cold water maintained at 5°C. The temperature for the cold shock was maintained at the desired value by either the addition of ice or water at room temperature, as needed from the readings of the attached thermometers. After the shock treatment, the different treatments were transferred with the mesh net to the already labelled aquarium (80 × 60 × 40 cm3) tanks connected to a re-circulatory system where they were incubated until hatching was observed. The percentages of eggs fertilized and hatched were then determined shortly after incubation began and at the end of incubation (24–27 h later) respectively using the equations given by Okomoda et al. (Reference Okomoda, Koh and Shahreza2018a, Reference Okomoda, Koh, Hassan, Amornsakun and Shahreza2018b) as shown below:
$$\% \;{\rm{ fertilization = }}{{Fertilized\,eggs\,in\,the\,Petri\,dish} \over {Total\,number\,of\,eggs\,in\,the\,Petri\,dish}} \times 100$$
$$\hskip-46pt\% \;{\rm{hatchability{\rm{ }} = {\it{{no.\,of\,hatched\,larvae} \over {total\,no.\,of\,spawned\,eggs}}} \times 100$$
The percentage abnormality and survival rate was also determined, respectively, at hatching and after 1 month of rearing, just before the determination of the fish’s ploidy status.
Confirmation of ploidy status
Upon larvae hatching and determination of breeding performance, the progeny from each treatment group were reared for the first month in triplicate in separate aquarium tanks (measuring 5 × 5 × 4 m3), under similar laboratory conditions. Fish were first fed for 2 weeks with freshly hatched Artemia nauplii ad libitum after absorption of their yolk. Subsequently, a commercial starter diet (with 45% CP) was used. To confirm the ploidy status of the fish, cytogenetic characterization was carried out using 1-month-old fingerlings and following the optimized method reported by Okomoda et al. (Reference Okomoda, Koh, Hassan, Amornsakun, Moh and Shahreza2018c) for the same species. In brief, fish were injected with 0.05% colchicine and allowed to swim for 3 h. Next, part of the gill tissue was removed and made into a cell suspension using 0.075 M KCl. After 1 h of exposure, the cell suspension was treated with Carnoy’s fixative three times (each treatment lasting for 20 min) before being aged for 3 days and fixed on a slide. Slides were stained with 10% Giemsa (1 h). Karyotyping Video Test Software (v.3.1) was then used for chromosome identification and counting. The diploid percentage of the progenies was determined and recorded for each treatment as the proportion of diploid to the total number of surviving progeny. It was also important to state that ploidy confirmation was only attempted in treatment with at least 10 surviving progeny at the time of characterization; this was to improve the accuracy of judgement of the successful induction of diploid gynogens and avoid any bias that would have characterized insufficient sample numbers.
Data analysis
Data from this study were analyzed using Minitab 14 computer software. Descriptive statistics were initially calculated for breeding parameters such as fertilization, hatchability, abnormality, survival and ploidy percentages. Then, the means of data from the treatment groups were separated using Fisher’s least significant difference test, when the assumption of normality and homogeneity were upheld. Alternatively, a non-parametric test (Kruskal–Wallis test) was used instead.
Results and discussion
Androgenetic induction aims at completely removing all chromosomes in the egg nucleus to prevent any maternal genetic contribution to the zygotic nucleus (Zhou et al., Reference Zhou, Wang, Liu, Chen, Sun, Zheng, Mahboob, Xu, Jiang, Zhuang and Jin2019). This study has shown that UV irradiation is effective for androgenetic induction with performance affected by both distance and duration of exposure (Table 2). Different irradiation methods have been applied to genetically inactivate egg nuclei. The earliest protocols used gamma irradiation, which has been proven effective in masu salmon Oncorhynchus masou, rainbow trout O. mykiss and brook trout Salvelinus fontinalus induction (Arai et al., Reference Arai, Onozato and Yamazaki1979; Parsons and Thorgaard, Reference Parsons and Thorgaard1984, Reference Parsons and Thorgaard1985; May et al., Reference May, Henley, Krueger and Gloss1988). UV irradiation has also been applied to European sea bass Dicentrarchus labrax (Colléter et al., Reference Colléter, Penman, Lallement, Fauvel, Hanebrekke, Osvik, Eilertsen, D’Cotta, Chatain and Peruzzi2014), Nile tilapia Oreochromis niloticus, Siberian sturgeon Acipenser baerii, Sterlet sturgeon Acipenser ruthenus (Balashov et al., Reference Balashov, Vinogradov, Kovalev, Barmintseva, Recoubratsky and Grunina2017), African catfish C. gariepinus (Bongers et al., Reference Bongers, in ’t Veld, Abo-Hashema, Bremmer, Eding, Komen and Richter1994) and common tench Tinca tinca eggs (Nowosad et al., Reference Nowosad, Kucharczyk, Liszewski, Targońska and Kujawa2015). X-rays have also been used on zebrafish Danio rerio, while a 60Co source has been applied to goldfish Carassius auratus (Corley-Smith et al., Reference Corley-Smith, Lim and Brandhorst1996; Paschos et al., Reference Paschos, Natsis, Nathanailides, Kagalou and Kolettas2001).
There is a paucity of information about the effect of distance of exposure at different durations on the performances of irradiated eggs. Many earlier studies have optimized different irradiation intensities at fixed distances for the efficiency of egg irradiation (e.g. Bongers et al., Reference Bongers, in ’t Veld, Abo-Hashema, Bremmer, Eding, Komen and Richter1994, Reference Bongers, Zandieh-Doulabi, Richter and Komen1999; Kirankumar and Pandian, Reference Kirankumar and Pandian2003; Christopher et al., Reference Christopher, Murugesan and Sukumaran2014). Although these studies were irradiation intensity-based, our current study suggested that the efficiency of irradiation received by treated eggs may be largely affected by distance and exposure time used. To improved the efficiency of irradiation, four UV lamps (i.e. pairs fixed on the dorsal and ventral sides) positioned at a distance of 27 cm from the point of egg irradiation were used in the study by Christopher et al. (Reference Christopher, Murugesan and Sukumaran2014). The recorded maximum diploid androgenesis was 21 and 14% at different cold-shock protocols (40 and 41°C, respectively) applied 30 min after activation of Heteropneustes fossilis irradiated eggs. Our study used just one UV source per irradiation chamber and recorded 100% diploid androgenesis in all surviving progeny whose eggs were exposed at 20 cm or less for various durations. The exposure distance of 30 cm was found to be ineffective for irradiation, irrespective of duration. This may have resulted from a reduction in irradiation intensity due to the large space between the UV bulb and eggs, as well as insufficient irradiation duration for the eggs to be effectively inactivated. However, it should be noted that performances may differ with different species. An earlier study by Balashov et al. (Reference Balashov, Vinogradov, Kovalev, Barmintseva, Recoubratsky and Grunina2017) reported that the complete genetic inactivation of ovicells of Siberian sturgeon A. baerii using UV irradiation was attained following a 120 s exposure, while it took 90 or 105 s to achieve the same effect in Sterlet sturgeon Acipenser ruthenus. The current study observed that irradiation at 20 cm for 1 min gave a moderately high hatchability and survival rate and efficiently induced diploid gynogenic progeny.
Although fertilization rate of eggs was high (i.e. 94–78%) in the irradiated groups, the following hatching was significantly low (Table 1). Okomoda et al. (Reference Okomoda, Koh and Shahreza2018a) hypothesized that values for fertilization and hatchability rates should be relatively close if fertilization was accurately determined in crosses with no post-zygotic isolation mechanism effects. However, this hypothesis did not account for the application of trauma to gametes or developing embryos through different chromosome manipulation techniques. Hassan et al. (Reference Hassan, Okomoda and Nurhayati2018a) stated that the shock protocol used for triploid induction in C. gariepinus was responsible for the significantly lower hatching rate compared with diploid groups. In our current study, however, the double trauma of irradiation and cold shock could have resulted in poor hatching of the diploid androgens (1–13%). Zhou et al. (Reference Zhou, Wang, Liu, Chen, Sun, Zheng, Mahboob, Xu, Jiang, Zhuang and Jin2019) found that conventional irradiation techniques for androgenesis do not only damage the egg nuclei but also its cytoplasmic organelles. According to Ocalewicz et al. (Reference Ocalewicz, Gurgul, Pawlina-Tyszko, Szmatoła, Jasielczuk, Bugno-Poniewierska and Dobosz2019), maternal RNAs responsible for the first cell cleavage in the fish embryos are sometimes damaged during ionizing radiation and therefore affect development after fertilization and influence hatchability negatively. Hatchability, as reported for androgenetic Heterobranchus longifilis in an earlier study by Olufeagba and Moses (Reference Olufeagba and Moses2011), was as low as 7%. Similar lower hatchability rates have been reported for stinging catfish, H. fossilis (Christopher et al., Reference Christopher, Murugesan and Sukumaran2012), African catfish, C. gariepinus (Bongers et al., Reference Bongers, Nguenga, Eding and Richter1995) and Ide, Leuciscus idus (Kucharczyk et al., Reference Kucharczyk, Targońska, Szczerbowski, Łuczyński, Rożek, Kujawa and Mamcarz2008) using irradiation accompanied by shock protocol.
The trend observed in this study suggested that higher distances improved hatchability rates, possibly because at these distances the intensity of irradiation received by the eggs was substantially reduced. However, prolonged exposure seemed to significantly reduce hatchability. Therefore, the Hertwig effect did not hold for the current study as, compared with decreased survival/hatchability from low doses of radiation and paradoxical higher recovery at higher doses, the reverse trend was observed in our study (Hertwig, Reference Hertwig1911; Christopher et al., Reference Christopher, Murugesan and Sukumaran2012, Reference Christopher, Murugesan and Sukumaran2014; Balashov et al., Reference Balashov, Vinogradov, Kovalev, Barmintseva, Recoubratsky and Grunina2017). Although earlier reported examples of the Hertwig effect in gynogenetic or androgenetic induction have been observed after irradiation with X-rays and gamma-rays, the finding by Kirankumar and Pandian (Reference Kirankumar and Pandian2003) also demonstrated a similar effect with UV ray inactivation of tiger barb Puntigrus tetrazona maternal genome. This, therefore, suggests that the Hertwig effect may differ between species, rather than be dictated by the different methods of irradiation.
High mortality rate characterized by androgenetic specimens is one of the limiting factors of commercial application of this technology in most cultured fish species (Ocalewicz et al., Reference Ocalewicz, Gurgul, Pawlina-Tyszko, Szmatoła, Jasielczuk, Bugno-Poniewierska and Dobosz2019). In the current study, the highest survival rate in successfully induced diploid androgenetic progeny was 36% (i.e. eggs irradiated at 20 cm for 1 min). According to Ocalewicz et al. (Reference Ocalewicz, Kuzminski, Pomianowski and Dobosz2013, Reference Ocalewicz, Gurgul, Pawlina-Tyszko, Szmatoła, Jasielczuk, Bugno-Poniewierska and Dobosz2019) and Michalik et al. (Reference Michalik, Dobosz, Wójcik, Zalewski and Ocalewicz2014, Reference Michalik, Dobosz, Zalewski, Sapota and Ocalewicz2015), the low survival of androgenetic progenies could be due to the expression of recessive alleles. According to Olufeagba and Moses (Reference Olufeagba and Moses2011), many of these abruptly increased paired recessive alleles are detrimental or lethal genes, and result in a reduced survival rate. The negative effect of homozygous loci for noxious recessive alleles has been linked to abnormalities observed in the study by Onozato (Reference Onozato1984) on coho salmon, Oncorhynchus keta. Similarly, deformities such as lordosis, scoliosis, kyphosis, c-shaped larvae and spiral larvae have been reported in androgenetic rainbow trout (Oncorhynchus mykiss) by Polonis et al. (Reference Polonis, Jagiełło, Dobosz, Rożyński, Kondraciuk, Gurgul, Szmatoła and Ocalewicz2019). However, many abnormalities observed in the current study were short tails (Figure 1) similar to that described as the ‘haploid syndrome’ in the study by Christopher et al. (Reference Christopher, Murugesan and Sukumaran2012, Reference Christopher, Murugesan and Sukumaran2014) on Heteropneustes fossilis. Hence, the impaired ability of the abnormal larvae to properly swim and capture live feed administered post endogenous feeding may have resulted in significant mortality. Although not characterized, the bulk of these abnormal progenies could be haploid androgens. According to Christopher et al. (Reference Christopher, Murugesan and Sukumaran2012), haploid androgens cannot survive through the processes of embryonic or larvae development, hence the mass mortality recorded.
Normal (upper) and abnormal (lower) (haploid syndrome) androgenic Clarias gariepinus.
Our study, therefore, observed an inverse trend between survival and abnormality rate, which translated to higher abnormalities that resulted in lower survival rates for androgenetic progenies and vice versa. Abnormalities such as triploid embryos have also been reported in C. gariepinus (Hassan et al., Reference Hassan, Okomoda and Nurhayati2018a), Black Sea turbot Psetta maxima eggs (Aydın and Okumus Reference Aydın and Okumus2017) and Anabas testudineus (Hassan et al., Reference Hassan, Okomoda and Sanusi2018b). However, the androgenic fish in this study received two different types of trauma compared with previous studies on triploid induction; the higher abnormality rate in the successfully induced androgenetic groups may be explained by higher levels of stress.
In conclusion, the findings of the current study showed that both the distance and duration of irradiation affected induction of androgenesis. While higher distances seemed to promote hatchability and survival, it was ineffective for induction of androgens. The optimal UV distance and duration that gave a better hatchability and survival rates, in addition to successfully inducing 100% diploid gynogenic progenies was 20 cm at 1 min. Future studies could test other means of induction that would reduce trauma infliction on eggs and which potentially reduce survival. This may include the use of cold shock to inhibit the extrusion of the second polar body in a normal developing diploid embryo, to allow expulsion with the egg pronucleus later. Another viable method of induction with less trauma could be irradiation of eggs, followed by fertilization with sperm from tetraploid C. gariepinus.
Acknowledgements
The authors are indebted to the management and staff of the Universiti Malaysia Terengganu, Malaysia in whose facility this research was conducted. We also acknowledge the help of the technical staff of AKUATROP during the experimental phase of this study. This study is part of the second author’s MSc thesis.
Ethical standards
Approval for the experimental protocols used in the study was given by the Universiti Malaysia Terengganu committee on research, therefore all methods used, as it pertains to animal care and use, were in tandem with specified international, national and institutional guidelines.
Conflict of interest
We declare that no fund was received for the conduct of this research; hence, we have no conflict of interest whatsoever (financial or otherwise).
Author contributions
HA, AAB, IM and OVT conceptualized and designed the study, NHJ experimented and collected needed data. OVT wrote the draft of the manuscript with help from SIO, OAS and AKI in preparing the text and table. All authors reviewed the manuscript and approved it for submission.
