Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(2): 23-30, 2023 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2112 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Gousia Jan, Asim Iqbal Bazaz, Azra Shah, Saima Andleeb, Irfan Ahmad, Durdana Qazi, Oyas Asimi, Bilal A. Bhat, Anayitullah Chesti (2023). Karyotypic analysis of Crucian carp, Carassius carassius (Linnaeus, 1758) from cold waters of Kashmir Himalayas. Caryologia 76(2): 23-30. doi: 10.36253/caryologia-2112 Received: April 14, 2023 Accepted: October 29, 2023 Published: December 31, 2023 Copyright: © 2023 Gousia Jan, Asim Iqbal Bazaz, Azra Shah, Saima Andleeb, Irfan Ahmad, Durdana Qazi, Oyas Asimi, Bilal A. Bhat, Anayitul- lah Chesti. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distrib- uted under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, pro- vided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Karyotypic analysis of Crucian carp, Carassius carassius (Linnaeus, 1758) from cold waters of Kashmir Himalayas Gousia Jan1, Asim Iqbal Bazaz2, Azra Shah1, Saima Andleeb1, Irfan Ahmad1,*, Durdana Qazi1, Oyas Asimi3, Bilal A. Bhat5, Anayitullah Chesti4 1 Division of Fish Genetics & Biotechnology, Faculty of Fisheries, SKUAST-K, Rangil, J&K, India-06 2 Division of Fisheries Resource Management, Faculty of Fisheries, SKUAST-K, Rangil, J&K, India-06 3 Division of Fish Nutrition & Biochemistry, Faculty of Fisheries, SKUAST-K, Rangil, J&K, India-06 4 Division of Aquaculture, Faculty of Fisheries, SKUAST-K, Rangil, J&K, India-06 5 Division of Social Sciences, Faculty of Fisheries, SKUAST-K, Rangil, J&K, India-06 *Corresponding author. E-mail: ahmadirfan@skuastkashmir.ac.in Abstract. Carassius carassius (Linnaeus, 1758) is an exotic fish to Kashmir, local- ly known as “gang gad” and commonly called as “crucian carp”. It belongs to family Cyprinidae. The present study aimed to identify the chromosome number of the Car- assius carassius and to optimize the colchicine concentrations (0.01%, 0.025%, and 0.05%) and hypotonic treatment timings (25, 35, and 45 minutes) for the chromosome preparation in Carassius carassius in order to obtain the highest number of clear and identifiable metaphasic chromosomal spreads. Data collected was analyzed and the means of each treatment was compared. The findings of the present study indicated that there was a significant influence of colchicine concentration, hypotonic timings as well as colchicine concentration× hypotonic timings (P<0.01) on the number of meta- phase chromosome spreads. Furthermore a significant (P<0.01) strong positive correla- tion obtained between colchicine concentrations, hypotonic timings and the number of metaphase chromosome spreads. The findings of the present study recommends further research into chromosomal modification techniques such as fish polyploid pro- duction, gynogenesis, androgenesis, and inter or intra-species hybridization is needed to generate unique and good inbred lines in aquaculture. Keywords: Carassius carassius, Colchicine, optimization, metaphase chromosome, kidney, hypotonic solution. INTRODUCTION The Cyprinidae (Teleostei: Cypriniformes) is a globally widespread and one of the richest and most vital family of fish (Al-Sabti, 1991; Kalbassi, 24 Gousia Jan et al. 2008). It is a large family of freshwater fishes common- ly called the carp family comprising of vast majority of bony fishes (Abdoli, 1999). The Crucian Carp (Car- assius carassius Linnaeus, 1758) is a medium-sized fish in the Cyprinidae family and is a widespread European species. Carassius carassius is locally known as “gang gad” and more specifically known as “crucian carp” and was introduced into Dal Lake in Kashmir, India between 1956 and 1958 (Shafi, 2012). It is one of the most abundant cyprinids in Dal Lake Kashmir and it has adapted to a wide range of habitats to cope with abiotic environmental challenges such as low oxygen levels and water temperature variations (Holopainen and Hyvarinain, 1985). The study of fish chromosomes has long been a pop- ular topic of study due to its usefulness in fish popula- tion conservation, cytotaxonomy, phylogeny research and evolutionary studies Luca et al. (2010); Bazaz et al. (2022). The development of techniques such as karyotyp- ing has made it possible to visualize undetected chromo- somal anomalies such as small portions of chromosomes and translocations of tiny parts of chromosomes to one another Bazaz et al. (2022). Fish cytology may be ben- eficial in interpreting the evolution of higher vertebrates and the interrelationships among the numerous divi- sions of fishes, because they are a primordial chordate group. In terms of species characterisation, evolution and systematics, chromosomal studies have acquired a lot of traction in recent years (Barat et al., 2002). One of the most critical parts of fish conservation is genetic characterisation. Using cytogenetic, biochemical-genetic and advanced molecular genetic approaches, this is con- ceivable. Cytogenetic characterisation is concerned with the study of chromosomes, which serve as genetic vehi- cles for a variety of genetic investigations, from the crea- tion of gene maps and chromosome organisation models to the investigation of gene function and dysfunction. Various methods are now being used to study fish chro- mosomes, with improvements and modifications such as the pretreatment with colchicine and hypotonic solution, which causes the cells to enlarge and the chromosomes to separate (Luca et al., 2010; Bazaz et al., 2022). There are a number of factors that may have con- tributed to the decline of fish species including habitat loss and degradation, displacement via competition with introduced species such as the polyploid biotype such as the goldfish (Carassius auratus) and the common carp (Cyprinus carpio communis). Moreover, all species of Carassius, including the crucian carp (Carassius caras- sius), Prussian carp (Carassius gibelio) and goldfish (Car- assius auratus) are often confused due to their morpho- logical similarity Knytl et al. (2021). Such confusion may lead to inappropriate stocking of wrong species instead of intended support of a population of crucian carp with negative consequences. Genetic contamination seems to be a very impor- tant but a hidden threat. Hybridization occurs and has been reported in various carassius species between such as Carassius carassius and Carassius gibelio (Prokes and Barus 1996). This type of hybridization was also confirmed using molecular (Wouters et al. 2012) and cytogenetic techniques (Knytl et al. 2013). Hybrids between Carassius carassius and Carassius auratus (Hanfling et al. 2005; Smartt 2007) and interge- neric hybrids between Carassius carassius and Cyprinus carpio (Hanfling et al. 2005) has been observed. Moreo- ver, molecular data suggest that these hybrids are able to reproduce and form relative generations by backcrossing (Hanfling et al. 2005, Wouters et al. 2012). The cytogenetics of Carassius carassius is still inad- equately understood. Interestingly, different diploid chromosome numbers 2n = 50 Raicu et al. (1981), 2n = 98 Manna, (1983), 2n = 100 Knytl et al. (2021) and 2n = 104 Chiarelli et al. (1969) has been reported. Similarly Knytl et al. 2021 reported the number of chromosomes in three Carassius species (C. auratus, C. carassius and C. gibelio) was uniform (2=100). The authors reported no differences in chromosomal morphology between male and female individuals. This findings confirmed homo- morphic sex chromosomes at least in diploid biotypes of the genus Carassius. Such an unclear condition encourages to present cytogenetic analyses of Carassius carassius with respect to hybridization processes and the related threats to indigenous species in Kashmir waters. Furthermore, the considerable karyotypic study on Carassius carassius has not been published in the Kashmir valley from dec- ades. The cyto-taxonomic status of Carassius carassius with respect to the species hybridization and diploid/ tetraploid status has remained unclear. In respect to its unclear distribution of possible diploid and/or tetraploid forms as well as hybridization process, the present study is an important contribution to the cytogenetics and cytotaxonomy of Carassius carassius. The present study deals with chromosomal characteristics of crucian carp (Carassius carassius) from the cold waters of Kashmir Himalayas. MATERIALS AND METHODS The research work was carried in the Division of Fish Genetics and Biotechnology, Faculty of Fisheries, SKUAST Kashmir, Rangil, Ganderbal, J&K, India. 25Karyotypic analysis of Crucian carp, Carassius carassius (Linnaeus, 1758) from cold waters of Kashmir Himalayas Sample collection A total of 45 specimens of Carassius carassius were collected from Dal Lake Srinagar, Kashmir and were transported live in an insulated box containing ice packs and equipped with aerators to the Fish Genetics and Biotechnology laboratory, faculty of fisheries, SKUAST- Kashmir. Fish were kept in 50L tubs equipped with con- tinuous aeration. Identification of Carassius carassius was done following the diagnostic characters described by Kullander et al. (1999). Chromosome preparation Chromosome preparation for Carassius carassius species was done by the methods described by Killger- man and Bloom (1977), and Shao et al. (2010) with some modifications. For each treatment, five samples were used. Three different concentrations of freshly prepared colchicine (0.01%, 0.025% and 0.05%), were injected intramuscularly at 1ml/100g of body weight for 90 min- utes to depress the mitotic division at the metaphase stage (Table 1). The fish were anesthetized using clove oil and were dissected. The anterior kidney was removed, homogenized and hypotonised simultaneously in 0.56% KCl for 25, 35 and 45 minutes at room temperature. Thereafter, the cell suspension was centrifuged for 10 min at 1300 rpm. Supernetant was removed and cells were fixed by chilled carnoy’s fixative solution (metha- nol: glacial acetic acid) and left overnight, fixation pro- cess was repeated two-three times until the clear suspen- sion was obtained with the fresh chilled fixative solution replaced every time. The suspension was centrifuged at 1300 rpm for 10 minutes before to each subsequent pro- cedure in the formal treatment; the supernatant was then removed, leaving one ml of the solution above the cell pellet in its place. The next solution was used to resuspend the cells. Slides were prepared by drop- ping method, three drops of the cell suspension on the pre-heated slide from about 40-50 cm height with slide slightly placed in an inclined position. The slides were stained using Giemsa stain (10%) distilled water and air-dried. Coverslips were placed on slides by mounting with D.P.X mountant to make them permanent and pro- tect them from drying out. Screening of slides The slides were observed under the field illuminated trinocular microscope (Olympus CX-21) fitted with cam- era at 100X objective and screened for good metaphase chromosomal spreads. Statistical analysis The data obtained from the different treatments was tested statistically by applying TWO-WAY ANOVA (SPSS, version 20) to evaluate the significant differences between each treatment and their interactions. Differ- ences among the means between the groups and within the groups were tested by “Duncan’s multiple mean range test” P<0.01. RESULTS The highest (Mean±SE) number of chromosomal spreads among the treatment groups were recorded in group T9 at 64.46±2.50 and the lowest (Mean±SE) num- ber of chromosomal spreads were recorded in group T1 at 1.93±1.83 (Table 2). A significant (P <0.01) positive correlation between colchicine concentration and the number of metaphasic chromosomal spreads at various hypotonic timings was observed as presented in Table 3 indicating that increase in colchicine concentration from 0.01% to 0.05%, the number of chromosomal spreads also increased (Table 3). Hypotonic treatment timings also estimated a significant (P<0.01) positive correlation with number of metaphasic chromosomal spreads (Table 4). The modal diploid chromosome number of C. caras- sius was found to be 2n = 100 (Figure 1). The chromo- somes were classified into 20 metacentric, 36 sub- meta- centric, 44 sub-telocentric and telocentric chromosomes 20 (M), 36 (SM), 44 (ST/T) (Figure 2). Figure 3 depicts the frequency percentage of dip- loid chromosome number ranging from 94 to 102 per Table 1. Experimental details. Treatments Colchicine concentration Hypotonic treatments timings (mins) No. of Samples/ treament No. of Slides / treatment T1 0.01% 25 5 15 T2 0.01% 35 5 15 T3 0.01% 45 5 15 T4 0.025% 25 5 15 T5 0.025% 35 5 15 T6 0.025% 45 5 15 T7 0.05% 25 5 15 T8 0.05% 35 5 15 T9 0.05% 45 5 15 26 Gousia Jan et al. metaphases. A total of 250.00 metaphasic chromosomal spreads studied from the cells of kidney tissues of Car- assius carassius revealed that the modal diploid chromo- some number of this species was found to be 2n = 100 which is valid over 70% of metaphasic cells. Figure 4 depicts the number of metaphase chromosome spreads at different colchicene concentrations (0.01%, 0.025%, 0.05%) and varied hypotonic treatment timing (25, 35, 45 minutes) indicating an increasing pattern of number of chromosome spreads with an increase in colchicene concentration and hypotonic treatment timings. DISSCUSSION Chromosomes are best Karyotyped during somatic metaphase whereby one can study chromosomal num- ber, size and morphology. To date, a variety of Karyo- typing techniques have been developed to visualise chromosomes of fish at various developmental stages, including tissue cultures (Lomax et al., 2000), squash- ing techniques (Armstrong and Jones 2003) and cell suspensions of tissues undergoing mitosis (Fan and Fox 1990; Henegariu et al., 2001). Nonetheless, approaches aimed at the embryonic and larval stages of fish have struggled to achieve a consistent number of recognis- able and widely distributed metaphase chromosomes, owing to differences in mitotic cell division rates among fish species (Shao et al., 2010). Pretreatment with colchicine combined with various kayotyping techniques has led to a revolution in fish chromosom- al studies (Roberts, 1964; Denton and Howell, 1969). Cochicine is a spindle poison used in traditional chro- Table 2. Average number of metaphase chromosome spreads among various treatments. Treatments Number of metaphasic chromosomal spreads (Mean±SE) T1 1.93±1.83 T2 3.6±2.84 T3 9.6±2.84 T4 26.13±2.55 T5 40.8±3.40 T6 47.86±2.32 T7 28.53±2.16 T8 50.93±1.79 T9 64.46±2.50 Table 3. Correlation between colchicine concentrations and num- ber of metaphase chromosome spreads. Colchicine concentrations (25 minutes of hypotonic timings) Colchicine concentrations (35 minutes of hypotonic timings) Colchicine concentrations (45 minutes of hypotonic timings) Metaphase Chromosome Spreads 0.890* 0.942* 0.969* Table 4. Correlation between Colchicine concentrations and num- ber of metaphase chromosome spreads. Hypotonic timings (0.01% Colchicine concentration) Hypotonic timings (0.025% Colchicine concentration) Hypotonic timings (0.05% Colchicine concentration) Metaphase Chromosome Spreads 0.761* 0.939* 0.980* * Significant at 0.01 level of significance. A B Figure 1. (A&B): Metaphase chromosome spreads of Carassius car- assius (2n=100) at 100x oil emulsion. 27Karyotypic analysis of Crucian carp, Carassius carassius (Linnaeus, 1758) from cold waters of Kashmir Himalayas mosomal preparation techniques to stop the cells from entering metaphase (Kligerman and Bloom, 1977). It is a naturally occurring alkaloid found in the Colchicum autumnale plant which binds to tubulin, preventing it from polymerizing, affecting microtubule dynam- ics and causing mitosis to be disrupted, generating a shift in the tubulin dimer, which is largely irrevers- ible. When a tubulin dimer bounds to colchicine the polymer becomes unstable and disassembles. (Schliwa 1986). Cells or larvae must be treated in a hypotonic solution after mitotic spindle inhibition to expand the nuclei and disperse the chromosomes over the slides (Moore and Best, 2001). Pretreatment with colchicine solution two hours before collection of the tissue in hypotonic sodium citrate or potassium chloride, fol- lowed by bringing the cells in suspension, fixation of cells in Cornoy’s fixative solution, smear on slides. and staining in various stains is now widely used for Karyotypic preparation. The staining techniques (e.g., aceto-orcein, haematoxylin, Giemsa, Wright and Leish- man stains) or banding techniques (e.g., Q-banding, G-banding, R-banding, C-banding and high-resolution banding) are used to stain chromosomes for various purposes (Calado et al., 2013; Moore and Best 2001). The choice of a right concentration and duration of exposure of colchicine is very important. This is because in sufficient amount of could fail to arrest the target cells amount could fail to arrest the target cells at metaphase stage (Rieder & Palazzo, 1992; Caperta et al., 2006), however too high a concentrations or prolonged expo- sure, on the other hand, may lead to chromosomal con- densation. As a result, the first stage in this technique was to optimize colchicine concentration to inhibit cell division in Carassius carassius. The optimum concen- tration of 0.05% for colchicine recorded in this study is similar to the findings of Karami et al. (2015), the authors reported that the optimum concentration of col- chicine was observed at 0.05% among the 0.01%, 0.025% and 0.05% tested in African catfish (Clarias gariepinus) and the zebrafish (Danio rerio). Similarly, Okomoda et al. (2018) reported that juveniles of Pangasianodon hypophthalmus and Clarias gariepinus had better chro- mosome spreads at 0.05% colchicines concentration compared to 0.01% and 0.025%. This, however, contra- M M SM SM SM ST/T ST/T ST/T ST/T Figure 2. Chromosomal classification of diploid Carassius carassius 2n= 100. Metacentric (M)= 20, Sub-metacentric (SM)=36, Sub-telo- centric/ Telocentric (ST/T)= 44. 4.8 6 16 70 3.2 0 10 20 30 40 50 60 70 80 94 96 98 100 102 Fr eq ue nc y % Diploid chrmosome number Figure 3. Frequency percentage of diploid Chromosome number of Carassius carassius. a c d a e g b f h 0 10 20 30 40 50 60 70 0,01% 0,025% 0,05% N o. o f m et ap ha se c hr om os om e Sp re ad s Colchicine Concenteration 25 minutes 35 minutes 45 minutes Figure 4. Graphical representation of number of metaphase chro- mosome spreads at different colchicene concentrations and varied hypotonic treatment timing. Data shown are mean ± SE with differ- ent letters are significantly different from each other (P≤ 0.01). 28 Gousia Jan et al. dicts with the findings of Shao et al. (2010) and Pradeep et al. (2011) in chromosomal preparation of fish larvae and reported the best metaphase chromosomal spreads at colchicine concentrations of 0.02% and 0.01%, respec- tively. While, this may be a sufficient amount concentra- tion to penetrate the vitelline membrane of the egg and the thin walls of the larvae, the present study shows this is not optimum for adult Carassius carassius. It’s vital to choose a suitable hypotonic solu- tion to swell the mitotic cell nuclei to the point of bursting and spread out the chromosomes after mitotic spindle inhibition (Moore & Best, 2001). Choosing an improper hypotonic solution and incubation period may result in overlapping or significant loss of chro- mosomes (Baksi and Means, 1988). Hypotonic treat- ment allows the swelling of the cell, which facilitates cell disruption and the dispersion of chromosomes when the cell contents are spread on slides. Potassium chloride (KCl 0.075 M) is one of the most commonly used hypotonic solutions in chromosomal prepara- tion protocols Bayat and Woznicki, (2006); Shao et al. (2010); Pradeep et al. (2011); Bazaz et al. (2022). The efficiency of potassium chloride (KCl 0.075M) over distilled water was demonstrated by Okomoda et al. (2018) reported that the number of clear metaphase chromosome spreads were significantly higher using the former than the latter. Smilarly, Ida et al. (1978) reported that the use of potassium chloride showed the best chromosome spreads as compared to other two hypotonic solutions of sodium citrate and dis- tilled water. In order to improve chromosome spreads, hypotonic treatment is essential. Hypotonic treatment induces swelling when cell contents are distributed on slides, facilitating cell disintegration and chromosom- al dispersion. To procure a desirable number of clear chromosome spreads, the hypotonic solution should be modified according to the species of fish and/or lar- val age (Karami et al., 2015). The current study found that hypotonic treatment time of 45 minutes was more effective than 25 and 35 minutes, however majority of the chromosomes were overlapped at 25 minutes of hypotonic treatment. In rainbow trout, chromosomal spreading was insufficient at 0.56% KCl for hypoton- ic treatment at a lower temperature Chourrout and Happe (1986). Furthermore, it was shown that the same concentration of KCl yielded superior results when the experiments were carried out at ambient temperature. However, 0.56% KCl for hypotonic treat- ment with an adequate time at room temperature pro- vided good results in the current investigation. Chromosome counts have long been used to char- acterize an organism or lineages most basic genetic characteristics. Chromosome counts have been utilized as useful phylogenetic indicators (Guerra 2008) and their role in evolutionary processes has been discussed several times (Mayr 1982; Clark and Donoghue, 2018). Cyprinid fishes have long been the considerable cytoge- netic research, with a focus on evolutionary issues (Taki et al., 1977). The 2n number in the Cyprinidae fam- ily ranges from 44 to 100 (Arai, 1982). The most com- mon diploid number in the Cyprinidae family is 50, which is regarded as the modal number in this family (Manna, 1984; Rishi, 1989). Cyprinid fishes have short chromosomes with centromere placements that range from median to nearly terminal, making it difficult to allocate some chromosomes to specific chromosomal groups as a result, making accurate identification of individual chromosomes nearly impossible (Rab and Collares-Pereira, 1995). The results of the current study revealed that Carassius carassius (crucian carp) possess 2n=100 chromosomes in its somatic cells. The results of the current study are in agreement with findings of Spoz et al. (2014); Knytl et al. (2013); Boron et al. (2010); Wang et al. (1995); Kasama and Kobayasi (1991); Mayr et al. (1986); Sofradzija et al. (1978); Hafez et al. (1978); and Kobayasi et al. (1970), as they all reported the same diploid chromosome count of 2n=100. However, Raicu et al. (1981) reported the diploid chromosome number 2n = 50 in Danube Delta of Carassius carassius indi- viduals. In contrast to Raicu et al. (1981), Chiarelli et al. (1969) reported a diploid chromosomal number of 2n=104, which might most likely be attributed to prepa- ration artifact. CONCLUSION The results obtained from the present study showed that 0.05% colchicine concentration resulted highest number of chromosomal spreads compared to other colchicine concentrations. 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Proceedings of Japan Academy 53(B): 231-235. Wouters J, Janson S, Lusková V, Olsén K.H. (2012). Molecular identification of hybrids of the invasive gibel carp Carassius auratus gibelio and crucian carp Carassius carassius in Swedish waters. Journal of Fish Biology 80(7): 2595-2604. doi: 10.1111/j.1095- 8649.2012.03312.x Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 76, Issue 2 - 2023 Firenze University Press Molecular classification of Barbeyaceae (Barbeya oleoides Schweinf.) using four different DNA barcodes Fatima Omari Alzahrani, Sami Asir Al-Robai Mitotic metaphase karyotype of the mosquito Anopheles arabiensis Patton (Diptera: Culicidae) from Kassala State, eastern Sudan Asma Mahmoud Hamza1,*, Sumaya Hussein Elboshra2 Karyotypic analysis of Crucian carp, Carassius carassius (Linnaeus, 1758) from cold waters of Kashmir Himalayas Gousia Jan1, Asim Iqbal Bazaz2, Azra Shah1, Saima Andleeb1, Irfan Ahmad1,*, Durdana Qazi1, Oyas Asimi3, Bilal A. Bhat5, Anayitullah Chesti4 Unfolding chromosomal uniqueness of the Scilloid ornamental Albuca virens by application of EMA based Giemsa- DAPI staining Biplab Kumar Bhowmick*, Sayani Nag Cytogenetic analysis of sympatric Trachelyopterus Valenciennes 1840 (Siluriformes, Auchenipteridae) species reveals highly conserved karyotypes despite the geographic distance Denise Felicetti1, Chrystian Aparecido Grillo Haerter2, Lucas Baumgärtner1, Leonardo Marcel Paiz1, Daniel Rodrigues Blanco3, Eliana Feldberg2, Vladimir Pavan Margarido1, Maelin da Silva4, Roberto Laridondo Lui1,* Cytogenetics of Diphysa americana (Mill.) M.Sousa (Leguminosae-Papilionoideae-dalbergioid clade), a rare species from the coast of Oaxaca, Mexico Fernando Tapia-Pastrana Cytogenetical studies of some Convolvulaceae members from the Western Ghats, India reveal uniformity in karyotypes R.N. Chougule, P.V. Deshmukh, P.E. Shelke, V.J. Patil, M.M. Lekhak* In vitro cytotoxic activity of phytosynthesized silver nanoparticles using Clematis vitalba L. (Ranunculaceae) aqueous decoction Nicoleta Anca Şuţan1, Diana Ionela Popescu (Stegarus)2, Oana Alexandra Drăghiceanu1, Carmen Topală3, Claudiu Şuţan3,*, Aurelian Denis Negrea4, Denisa Ştefania Vîlcoci4, Georgiana Cîrstea4, Sorin Georgian Moga4, Liliana Cristina Soare1