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Triploidy induction which produces sterility in tilapia is an interesting option which has considerable potential in advanced aquaculture practices (Penman et al., 1987; Mair and Little, 1991; Mair, 1993). Triploidization results in an addition of extra set of chromosome by retention of second polar body causing an increase in cell and nuclear size (Graham et al., 1985; Benfey, 1991; Stillwell and Benfey, 1996; Benfey, 1999 and Hyndman et al., 2003). Because of this extra set of chromosome, the cells of triploids maintain the nucleo-cytoplasmic ratio, therefore, the cells of most of the organs (brain, retina, kidney, liver, testis, ovaries) and tissues (blood, cartilages, muscles, epithelia) are larger than those of their diploid counterparts (Benfey, 1999). However, in order to compensate the increased cell size and nucleus, the organs and tissues of triploid individuals have to reduce the number of cells to maintain its morphology similar to diploids (Maxime, 2008). The exact scientific explanation, however, for this reduced cell number is not yet understood.
A variety of direct detection techniques have been used for differentiating the normal and ploidy induced fishes namely; DNA staining and fluorescence quantification of DNA with flow-cytometry micro-photometry, micro-densitometry, mechanical particle size differentiation; silver staining of nucleolar organizer regions and cytological karyotyping. However, each technique has its own advantages and disadvantages, varying from technical expertise, accuracy and expensive equipments, etc. are required for such techniques. Direct determination method by cytological karyotyping is the most irrefutable method of determining polyploidy among all these existing methods (Maxime, 2008). The flow-cytometry is the fastest and most accurate technique available among all known methods to determine the ploidy in fishes (Maxime, 2008). The other alternative indirect and easiest method to determine ploidy is by checking the cells or nucleus size of erythrocytes (Purdom, 1993; Tiwary et al., 2004). A number of studies demonstrated that the erythrocyte and nuclear dimensions in triploid fish varied significantly from those of diploid fish. As the technique of erythrocyte measurement is simple, rapid and inexpensive, it is one of the most acceptable techniques. Other alternative indirect methods also include coulter counter analysis, erythrocyte measurement of the main axis length from computer-assisted image analysis and staining of erythrocyte using ¬‚uorescent nuclear stain (Maxime, 2008).
The nuclear major axis measurement of erythrocyte can readily and widely be used for identifying triploids and also tetraploids in tilapia (Penman et al., 1987). Erythrocyte nuclear measurement has been used to confirm ploidy status of transgenic tilapia, O. niloticus where the erythrocytes of triploids is 1.5 times larger than diploids (Razak et al., 1999). Varadaraj and Pandian (1990) used RBC nuclear volume for detection of diploid and triploid fingerlings while producing all female sterile triploid in O. mossambicus. They recorded an increase in nuclear size near to 1.5 times greater than that of the diploid. Hussain et al. (1995) used erythrocyte nuclear major axis from the blood of adult fishes of O. niloticus for discriminating triploid and diploid individuals.
A few consistent difference might occur among the hematological characteristics of triploids fishes which are namely; (1) larger erythrocytes (2) increased hemoglobin content and (3) lesser erythrocyte counts than erythrocyte counts of diploid (Benfey, 1999 and Cogswell et al., 2002). This increase in cell size especially of erythrocytes helps in oxygen carrying capacity from the external medium to the cell which might compromise the ability of triploids to utilize more oxygen (Cal et al., 2005). The increased cellular volume in red blood cells of triploids is geometrically associated with a decrease in the surface area to volume ratio (Cogswell et al., 2002). The erythrocytes of triploid individuals exhibit peculiar morphological characteristics with much higher frequencies in comparison to diploids such as; attenuated cell shape and segmented nuclei (Piercy, 2005).
A detailed investigation has been carried out by Pradeep (2010) to detect the triploids in red hybrid tilapia. For his study, he collected a total number of 60 larvae, 30 larvae produced by heat shock treatment (temperature 41o C) applied for duration of 3.5 minutes and 4 minutes after the fertilization of eggs. A group of 30 larvae without applying any shock treatment (control groups) were reared separately under identical water conditions in aquariums tanks. The blood of actively swimming larvae (25 g in weight) from both groups was collected to study the erythrocyte. Twenty specimens each from the control and the heat shock treated group of similar size were randomly selected for the collection of blood. The identification of the ploidy level of each fish using well spread metaphase stages was also simultaneously carried out to compare the results in both groups. For this, each fish was given a dose of 0.01% colchicines at a rate of 1 ml/100 g administered with the help of a hypodermic syringe at the dorsal fin, just above to the lateral line of the fish. This was followed by keeping fishes inside an aerated aquarium tanks (120 l) for 4 hours.
Differences in erythrocyte measurements between diploid and triploid of tilapia
Preparation of blood smears for erythrocyte measurements
A fish at first was severed few millimeters anterior to the caudal peduncle to cut the caudal vein by a pair of sharp scissors for the collection of blood. A drop of blood was then taken from the cut region and placed on a clean microscopic slide and gently smeared using a cover slip. Slides were allowed to dry for few seconds and then fixed in 95% methanol. The slides were stained with Giemsa stain (10%) for 20 minutes after proper drying and mounted by DPX and a cover slip, simultaneously. Length and width of the cell and nucleus were measured by a micrometer for 25 erythrocytes from each group of fishes. As erythrocytes of tilapia are ellipsoid in shape, the cell and nucleus volumes are calculated by an equation; V=4/3Ï€ab2 where a and b are the major and minor semi-axis of the cell and nucleus (Uzunova, 2002). The cytoplasmatic volume was also calculated by subtracting nucleus volume from the mean erythrocyte cell volume whereas surface area of the erythrocyte cells and their nucleus was calculated by an equation S=abÏ€/4 (Dorafshan et al., 2008). Photographs (1000X magnifications) of erythrocytes collected from both the groups were taken for further studies.
Chromosome preparation for detection of ploidy level
The same fish was used for chromosome preparation to find out the ploidy level immediately after the collection of the blood for erythrocyte study (Sofy et al., 2008). The fishes were killed by pithing near to the brain, their kidney was removed and used for chromosome preparation. The kidney at first was washed properly in an isotonic solution of NaCl (0.7%) to remove excess blood and debris. NaCl was also used to remove tissues which were finely chopped using a sharp blade. The chopped tissues from the petri dishes were then transferred to small plastic vials and then homogenized for a minute that was followed by their further transfer to a bigger centrifuge tube (15 ml capacity). Tissues were then hypotonised with 8 ml KCl (0.56% kept at room temperature) which was added to each centrifuge tube. Hypotonic treatment was given for a total duration of 40 minutes and then the solution changed two times at 15 minutes and 30 minutes of intervals. The resultant solution was then centrifuged at 100 x g for 7 minutes and the supernatant removed carefully. The tissues were immediately fixed in the same tube with 8 ml cold Carnoy's solution (3:1) for 30 minutes. The solution was again centrifuged at 100 x g for 10 minutes after the fixation time and the supernatant was removed and re-fixed in Carnoy's solution for 10 minutes at 4o C. The centrifugation and re-fixation process was repeated again for third time after one hour before fixation. After completing the fixation, the cell suspension and tissues were taken and placed on a clean microscopic slide. Tissues were chopped thoroughly to get white suspension using a sharp scalpel. A drop of distilled water was put onto the tissue to prevent drying and for proper dissociation during chopping. Carnoy's solution (30 µl) was put onto the chopped suspension to facilitate proper spreading of cells on the slide. Cells were then spread using the edge of another microscopic slide. Immediately, the slide with spread cells was warmed under the flame using an alcohol lamp until complete evaporation of liquid. The slide was then air dried for 10 to 15 minutes and later rinsed in acetone solution to remove the oil droplets. These slides were again air dried for 10 to 15 minutes and then stained with freshly prepared 10% Giemsa stain (prepared in 0.01M phosphate buffer; pH=7) for a period of 30 minutes. Slides were finally rinsed in distilled water, air dried and mounted with DPX after 10 minutes of xylene wash. The metaphase spreads were photographed and number of chromosome spreads was counted by observing slides under 400X and 1000X (oil immersion). The maximum number of chromosome spreads as much as possible was counted for ploidy determination.
In control groups where no shock treatment was used, all individuals were diploid (Pradeep, 2010). However, in fishes where heat shocked treatment was given for triploid induction, 20 out of 22 fishes were triploids. The karyotyping analysis of diploid fishes showed the presence of 44 numbers of chromosomes whereas 66 in triploid fishes. A significant difference in cell and nucleus major axis, minor axis, volume, surface area and cytoplasmatic volume was observed between diploid and triploid erythrocytes. The different measurements of erythrocyte of diploid and triploid red hybrid tilapia are summarized in Tables 9.1 & 9.2.
In diploid, the mean values of cell major axis, cell minor axis and cell surface area were; 10.43±0.51; 6.69±0.36 µm and 54.70±4.05 µm2, respectively (Table 9.1). The mean values of nucleus major axis, nucleus minor axis and nucleus surface area were; 4.47±0.34; 2.50±0.25 µm and 9.08±1.45 µm2, respectively. Cell volume in diploid fishes was 245.56±29.39 µm3 whereas nucleus volume 15.89±4.25 µm3. The cytoplasmatic volume in diploid was 229.68±26.95 µm3 (Table 9.2). Similarly in triploid fishes, the mean values of cell major axis, cell minor axis and cell surface area were; 13.32±0.37; 7.46±0.44 µm and 77.70±6.28 µm2, respectively (Table 9.1). The mean values for nucleus major axis, minor axis and surface area, were; 5.89±0.38, 2.93±0.34 µm and 13.66±2.29 µm2, respectively. The cell volume in triploid fishes was 390.67±51.69 µm3 whereas the nucleus volume was 26.78±6.53 µm3. In triploids the cytoplasmatic volume was 362.81±47.60 µm3 (Table 9.2).
The increments in cell major axis and minor axis were; 27.7 and 11.5%, respectively, higher in the cells of triploid fishes as compared to cells of diploid fish. The ellipsoidal size of erythrocytes in triploid fishes was relatively larger as compared to the diploids. The increase in nucleus size in triploid was also greater by 31.7% for the major axis as compared to minor axis by 17.2%. Similarly, the increase of nucleus cell surface and volume were; 50.4 and 68.5%, respectively, as compared to cell surface area (42%) and cell volume (59%) in triploids. Cytoplasmatic volume of the cell was increased by 57.9% in triploids as compared to diploid (Table 9.2).
Table 9.1: Cell and nucleus size and their combined parameters of the diploid and triploid red hybrid tilapia (mean ± SD)
[Reproduced from Pradeep (2010)]
Cell major axis (µm)
10.43 ± 0.51
13.32 ± 0.37
Cell minor axis (µm)
6.69 ± 0.36
7.46 ± 0.44
Cell surface area (µm2)
54.70 ± 4.05
77.70 ± 6.28
Nucleus major axis (µm)
4.47 ± 0.34
5.89 ± 0.38
Nucleus minor axis (µm)
2.50 ± 0.25
2.93 ± 0.34
Nucleus surface area (µm2)
9.08 ± 1.45
13.66 ± 2.29
(n= number of specimens)
Table 9.2: Cell, nucleus and cytoplasmatic volume of the diploid and triploid red hybrid
tilapia (mean ± SD)
[Reproduced from Pradeep (2010)]
245.56 ± 29.39
390.67 ± 51.69
15.89 ± 4.25
26.78 ± 6.53
229.68 ± 26.95
362.81 ± 47.60
(n= number of specimens)
The parameters such as; nuclear and cytoplasimc volumes and nuclear surface area though showed higher values in triploid as compared to the diploid fishes and it could be considered for determining the diploid and triploid fishes. But determination of these parameters following the same technique is cumbersome and not easy to use. However, in O. mossambicus, Varadaraj and Pandian (1990) used RBC nuclear volume for discriminating the diploid and triploid fingerlings where they found an increase in nuclear size of 1.5 times greater than that of the diploid. In other study conducted by Lincoln and Scott (1983) on rainbow trout, the nuclear volume of RBC has been used for determining the ploidy in triploid. However, in triploid of red hybrid tilapia, it has been 1.68 times greater as observed by (Pradeep, 2010). The nuclear volume ratio was 1:1.68 as compared to the values (1:1.86) obtained in Misgurnus anguillicaudatus (Gao et al., 2007). An increase in nuclear volume by 87% has been observed by Dorafshan et al. (2008) as compared to the theoretical expected 50% increase. The nuclear volume has been found to increase by 68.5% in triploids (Pradeep, 2010). The erythrocyte volume of 45.9% has been reported almost reaching close to the expected theoretical 50% increase in sea bass (Dorafshan et al., 2008) whereas in red hybrid tilapia the increment of erythrocyte volume has been 59% (Pradeep, 2010).
The major and minor nuclear axis were the other parameters which showed relatively the higher values in triploid as compared to the diploid fishes in red hybrid tilapia (Pradeep, 2010). As such these parameters could easily be used to differentiate the diploid and triploid fishes. Penman et al. (1987) has also recommended nuclear major axis measurements of erythrocyte of tilapia for the identification of triploid and tetraploid fishes. Razak et al. (1999) demonstrated the ploidy status of transgenic tilapia, O. niloticus using erythrocyte nuclear major and minor axis measurements. In O. niloticus, the measurement of erythrocyte nuclear major axis has been found to be an appropriate option for identifying the triploid and diploid individuals as reported by Hussain et al. (1995).
In triploid of red hybrid tilapia, the cellular major axis is 1.27 times greater than the diploid (Pradeep, 2010) whereas in triploid sea bass it has been 1.2-1.3 times (Felip et al., 1999). Cell and nuclear major axis, nuclear and cell volume and cell minor axis were greatly different in diploid and triploid sea bass. They suggested that the most appropriate parameter to differentiate diploid and triploid is the major cell axis and cell volume. The cellular major axis in Caspian salmon has been reported 27% higher than the minor axis (22%), thus making the cells ellipsoidal in shape (Dorafshan et al., 2008). Similar result has been obtained in red hybrid tilapia where the shape of the cells is ellipsoidal in triploids (Pradeep, 2010). The cellular and nuclear length and width have been found to be 25 and 20% larger whereas nuclear length and width 21 and 18% greater in erythrocytes of triploid than diploids in shortnose sturgeon (Beyea et al., 2005).
The ellipsoidal size of erythrocytes in triploid fishes is being relatively larger as compared to the diploids. However, the morphology of erythrocytes and their nuclei in triploids of common carps has been found to change from round to ellipsoidal in triploid as the major axis of the erythrocytes enlarged more than the minor axis, considerably (Ueno, 1984). In brook trout, Salvelinus fontinalis, the increase in size of erythrocyte nucleus with increase in number of chromosome has been observed where the major axis is being recommended as the simplest method for ploidy identification (Woznicki and Kuzminski, 2002). Cherfas et al. (1994) also showed a change in size and shape of erythrocytes and their nuclei and used these parameters in determining the ploidy level in common carp (Cyprinus carpio) with a less than 4% error.
The erythrocytes of triploid are 1.5 times greater than diploids fishes. The volume of nucleus in triploids of red hybrid tilapia has also been found to increase by 68.5% than the diploids (Razak et al., 1999). The cytoplasmic volume ratio in triploid red hybrid tilapia is 1:1.57 whereas 1:1.48 in loach, Misgurnus anguillicaudatus (Gao et al., 2007). The cytoplasmic volume in triploid red hybrid tilapia has been 57.9% higher than the diploid fish (Pradeep, 2010). Peruzzi et al. (2005) observed that the erythrocyte size of several indices including the increase in cytoplasmic surface area (32%) and nucleus (50%) however, a considerable decrease in erythrocyte number (34%) in sea bass, Dicentrarchus labrax was observed. The surface area of the cell and nucleus are; 1.42 and 1.50 times higher respectively in triploids of red hybrid tilapia (Pradeep, 2010) whereas in triploid of common carps, these values ranged to 1.44 and 1.40 times higher (Ueno, 1984). However, there was no significant effect of ploidy on erythrocyte nuclear minor axis (Svobodova et al., 1998).
Techniques of detection of ploidy by karyotyping
The triploidy induction study, way back 20th century, was done in several fishes and crustaceans motivated the researchers to modify several conventional chromosome preparation techniques. These modified techniques have mostly focused on detection of ploidy using embryonic tissues (Kligerman and Bloom, 1977; Chourrout and Itskovich, 1983; Chourrout and Happe, 1986; Don and Avtalion, 1986; Hussain and McAndrew, 1994). These researchers might have taken embryonic tissues for ploidy detection as the embryonic and larval stages are the most suitable stages for chromosomal preparations where cells multiply rapidly in soft tissues at these stages (Tan et al., 2004). Application of direct chromosome preparations is rather more desirable especially for small larvae from which blood cannot be collected. But the age or stage of larvae has a significant influence on the quality of the readable metaphase stages (Chourrout et al., 1986).
Cytogenetical studies in fish have valuable importance in evolutionary studies, taxonomy, mutagenesis and in aquaculture industries especially for ploidy determination and fish stock management (Grey et al., 1980; Chourrout et al., 1986; Fenocchio et al., 1988; Foresti et al., 1993; Demirak and Unlu, 2001). Advancement in teleost cytogenetical studies has been followed by a variety of karyotypic techniques including tissue cultures (Roberts, 1964), squashing of the testis (Roberts, 1964; Ohno, 1965), embryonic tissues or haematopoetic materials (Simon, 1963; Yamada, 1967), smearing of gill epithelium (Mcphail, 1966; Stewart, 1968), kidney (Ojima, 1972; Arai, 1973; Ueno, 1977) and air drying techniques (Eicher, 1966; Fukuoda, 1972; Bertollo et al., 1978; Thode et al., 1998) and colchicine treatment (Yamazaki, 1971). Karyotyping or chromosome counting is the analysis of chromosome number and morphology of a species. Chromosomes can be obtained from any eukaryotic organism whose cells are actively dividing. With the progress of genetical studies like chromosome set manipulation, cytogenetic studies have also gained popularity in aquaculture reseraches considerably. Among the artificial polyploidy techniques, triploidization has been considered as the most successful technology (Solar et al., 1984) and successfully executed in several commercially important species of fishes (Pandian et al., 1998; Khan et al., 2000).
In most of the previous studies, dropping method was used to disperse cells for karyotyping where tail region of larvae used for chromosomal preparation. Among several techniques employed to prepare fish chromosomes, air drying method of Evans et al. (1964) has been the most popular and widely adopted method. This method is the most recent one and used effectively for preparing animal chromosomes whereas the squash technique is the oldest and the most widely used method for spreading and flattening metaphase chromosomes (Denton, 1973). Although these methods gave some results, a large portion of cells has been found to be lost during the experiment as a result of dropping the cell from a height. More over this method requires more technical skill to drop cells exactly onto the top of a preheated slide. Some researchers have also attempted to drop cells from a height onto the frozen slides for preparing the chromosome spreads (Ojima et al., 1964; Ida et al., 1978; Lamatsch et al., 1998). Researchers even tried with water vapour from a water bath for heating the slides and spreading cells (Henegariu et al., 2001). Chourrout and Happe (1986) reported that the conventional techniques of chromosome preparation by air drying technique after colchicine injection in young fishes resulted in inadequate metaphase spreads (MacPhail and Jones, 1966; Kligerman and Bloom, 1977).
Observation of sex chromosomes from cytological preparations allows letting an accurate proofreading differentiation which is an accurate method for directly differentiating the diploid or triploid individuals with 2 and 3 sex chromosomes (Moreira-Filho et al., 1993; Devlin and Nagahama, 2002; Molina and Galetti, 2007; Vasconcelos, 2009). Karyological studies have given valuable information on the number, size and morphology of chromosomes which is essential for polyploidy manipulation in fish (Khan et al., 2000). Therefore, cytogenetic application or karyotyping has been considered as a vital tool for perfecting the stock enhancement techniques such as chromosomal manipulation, hybridization and other related genetic engineering techniques (Tan et al., 2004).
Preparation of chromosome spreads
In a study carried out by Pradeep (2010) karyotyping, tissues from one day old larvae of artificially bred red hybrid tilapia were collected for chromosomal spreads. The colchicines concentration was initially optimized to get the maximum metaphase spreads. For this series of trial experiment were conducted. For each experiment duplicate trial with twenty larvae per batch was considered. First trial was performed using larvae from one batch whereas the second on another batch of larvae. Three different concentrations of colchicine i.e. 0.005, 0.01 and 0.1% of 20 ml were taken in three separate petri dishes. Twenty larvae (one day old) for each concentration were taken and immersed into the colchicine solution. For each concentration, the duration of the colchicine treatment was also altered for 2, 4-6 and 10 hours. In all trial experiments, freshly prepared colchicine was used and samples were kept undisturbed at the room temperature. After desired durations of 2, 4-6 and 10 hours, all larvae from different batches of colchicine treatments were taken out from the petri dishes and transferred into an isotonic solution of 0.7% chilled sodium chloride separately, to anaesthetize larvae. The yolk sac and debris of larvae were carefully removed using a sharp scalpel. In continuation an attempt was further made to optimize the duration of hypotonization. For this the whole body was hypotonized in 0.56% potassium chloride (0.075 M) for durations of 20, 30, 40 and 50 minutes at the room temperature to find out the most effective hypotonic treatment.
Similarly, in order to find out the most appropriate ratio for Carnoy's solution, 3 different ratios of methanol: acetic acid (2:1, 3:1 and 4:1) of Carnoy's fixative was prepared and testified. The complete body of the larvae was then fixed in a small glass vial containing 15 ml of freshly prepared Carnoy's solution with different ratio and kept separately at 4o C. The fixative was initially changed twice at 20 minutes intervals in the same vial. After this these larvae were stored with fixative at 4o C for a minimum duration of 6 hours inside the same glass vial. Slides for chromosome preparation were cleaned thoroughly before their use. Slides and cover slips were kept in 95% ethanol overnight and then rinsed with distilled water (Yu et al., 1981; Sofy et al., 2008) followed by swabbing of the slides by soft tissue paper. After fixation, whole body of the larvae was taken out from the vial and on one slide one larva was taken for chopping. A drop of distilled water was put on the larva to prevent drying and for the proper dissociation of embryonic cells. Chopping method as suggested by Yamazaki et al. (1981) was then followed. The effectiveness of chopping method was also checked using both distilled water and 50% acetic acid as described by earlier workers.
The larva was then chopped thoroughly using a sharp scalpel to get white suspension. A 30 µl of Carnoy's solution was put onto the chopped suspension to facilitate proper spreading of the cells throughout the slide. Cells were then spread using one side of another microscopic slide. Immediately, the slide with spread cells was warmed under the flame using an alcohol lamp until the liquid evaporated copletely. Slides were then air dried for 10 to 15 minutes and later rinsed in acetone solution to remove the oil droplets. All slides were again air dried for 10 to 15 minutes and then stained with freshly prepared 10% Giemsa stain (prepared in 0.01M phosphate buffer of pH=7). Different concentrations of Giemsa stain (5, 10 and 20%) under altered timing of 10, 20 and 40 minutes were examined and compared. After staining, slides were dipped in xylene for 10 minutes. Following the xylene wash, slides were subsequently rinsed in distilled water, air dried and mounted with DPX. The metaphase spreads were photographed and number of chromosome spreads were counted under 400X and 1000X (oil immersion). As many chromosome spreads as possible were counted on each slide and compared to select the most appropriate parameters for getting the best and maximum chromosome spreads. However, chromosome spreads which were either too dispersed or clumped, excluded during the counting. The batch showing highest number of well metaphase spreads in all trial experiments was selected for further experiment.
The appropriate concentration of colchicine (0.01%), duration of colchicine treatment (4-6 hours), hypotonic treatments (for 40 minute), Carnoy's solution (methanol: acetic acid ratio 3:1) for fixation and concentration of Giemsa (10%) for 20 minutes duration; were considered for further experiments by Pradeep (2010). Results of this improved method showed that good chromosome spreads could be made from the whole body of red hybrid tilapia. The minimum and maximum number of chromosome spreads were; 13 and 25 for the diploid respectively whereas 11 and 21 for triploid respectively on one slide. In the present experiment the whole body was taken because of the fact that at embryonic stage of the fish, a very little transparent tissue was available for making the spread. Slides with diploid and triploid embryonic larvae showed a high percentage of chromosomal spreads ranging from 95-100% in all the three experimental attempts using different batches of the larvae. Secondly, the improved technique eliminated the drooping of cell suspension onto pre-heated or frozen slides. Instead, the chopping method also avoids the dropping of cell suspension where all steps of preparations are carried out only on one slide within a shorter duration.
For karyotypic analysis, every single step including the preparation of tissues and slides are critically important for obtaining large number of well spread metaphases. Generally, a cell culture population has a mixture of cells at all stages of the cell division cycles at any given time. However, chromosome preparations could only be possible when this cell culture population has enough mitotic cells. For enriching these mitotic cells, colchicine drugs are used which allowed arresting of chromosome division at a metaphase stage of the cell division. Optimum concentration of colchicine and the duration of treatments are critically important for getting better results (Pradeep, 2010). Thus the initial step in the technique is colchicine treatment of the organism for arresting the cell division. The optimum colchicine concentration for red hybrid tilapia fish larvae has been determined as 0.01% for 4-6 hours (Pradeep, 2010). Hussain et al. (1994) has already reported that inadequate or over concentration of both colchicine treatments can lead to many un-burst cells with uncountable and over lapping chromosomes in the slide preparation. Pradeep (2010) also showed many un-burst cells that resulted in clumsiness of cells quite often during changing in concentrations of colchicine (0.005% and 0.1%). Duration of 2 and 10 hour treatments showed limited chromosome spreads and more clumsy cells. Colchicines stored in a refrigerator at a temperature of 4o C has not been found as effective as freshly prepared colchicines solution.
Hypotonic treatment is an important and crucial factor in improving the chromosome spreads. This treatment helps in removal of lipids and denatures proteins. It also allows the swelling of the cell which facilitates cell disruption and the dispersion of chromosomes when the cell contents are spread on slides. Ida et al. (1974) reported that potassium chloride facilitated the best chromosome spreads as compared to sodium citrate and distilled water. The hypotonic treatment with potassium chloride has been standardized for 40 minutes where a comparison with treatment times for 20, 30, 40 and 50 minutes are used in red hybrid tilapia. A time of below 40 minutes resulted in more sub-burst cells whereas; chromosomes are found overlapping for 50 minutes. Chourrout and Happe (1986) reported that the chromosome spreading was insufficient with 0.56% KCL for hypotonic treatment in rainbow trout at the lower temperature. However, it showed slightly better results by performing the experiment at ambient temperature. According to the same author, trisodium citrate as hypotonic treatment gives significant improvement in chromosome spreading. However, 0.56% KCL treatment for hypotonization with appropriate duration at room temperature has been found to yield better results (Pradeep, 2010).
A fixative solution of Carnoy's at a ratio of 3:1 has been most effective as compared to both 2:1 (Ida et al., 1974) and 4:1 ratio (Hussain et al., 1994). The Carnoy's fixative allows in preserving the internal structure of the cells for better staining of the chromosome (Comings, 1978). The tissues can be stored in this Carnoy's solution for a longer duration of more than a month (Pradeep, 2010). In the technique improvised by Pradeep (2010) for red hybrid tilapia, the steps followed by Klingerman and Bloom (1977) and Hussain et al. (1994) of maceration and dissociation of cells with 50% and 60% acetic acid respectively were avoided. Distilled water (2-4 µl) has been used for preventing the cells from drying when kept onto slides for chopping (Pradeep, 2010). In the modified technique, different durations of staining along with different concentrations of Giemsa stain were also tried. A concentration of 5% Giemsa stain for its treatment of 20 minutes as described by Bayat and Woznicki (2006) was not very effective whereas, at a concentration of 20% as suggested by Don and Avtalion (1986), counting of the chromosome was difficult. The altered timing and concentrations of Giemsa stain affected significantly the visibility and brightness of the spreads on slides. A concentration of 10% Giemsa stain prepared in 0.01 M phosphate buffer of pH 7 for 20 minutes duration as described by Hussain et al. (1994) was also tried in red hybrid tilapia obtained clear images.