Triploidy determination in rainbow trout (Oncorhynchus mykiss) based on erythrocytes dimensions

Transcription

1 Triploidy determination in rainbow trout (Oncorhynchus mykiss) based on erythrocytes dimensions Ioan Bencsik 1, Nicolae Pacala 1, Gabi Dumitrescu 1, Dorel Dronca 1, Jana Stanculet 2, Liliana Petculescu-Ciochina 1 1 Banat's University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Science and Biotechnologies, , Calea Aradului Street, No. 119, Timisoara, Romania 2 SC Detergenti SA, , Calea Stan Vidrighin,No. 5,Timisoara, Romania Abstract Triploidy induction at fish is characterized by modification of normal diploid chromosome set (2n) into triploid set (3n). Experiments were carried out on biological material from rainbow trout (Oncorhynchus mikiss) during the natural breeding season. Polyploidy was induced by exposing the eggs to heat shock. Blood smear was used as a technical method, to determine diploid and triploid status. Staining of blood smear was performed by Pappenhein method. The erythrocytes area and perimeter measurements done comparatively on triploid and diploid individuals may represent an indicator to determine the ploidy level of individuals. Erythrocytes area (µm²) for triploid individuals is 1.76 times higher than at diploid individuals, and perimeter (µm²) 1.31 for triploid individuals is times higher than in diploid individuals. Keywords: erythrocytes, heat shock, rainbow trout, tetrapoidy 1. Introduction Aim of the study was to determine triploid individuals obtained after thermal shock application on fertilized rainbow trout eggs [1,2]. Triploidy on fish is characterized by changing the set normal diploid number (2n) of chromosomes in the state of triploidy (3n). Triploidy can be achieved by inducing thermal shock, by pressure shock, different ph, different concentrations of chemicals, etc. [3 9]. Polyploid cells have in addition one or more sets of chromosomes; therefore will have a larger nucleus. The ratio between the cytoplasm and the nucleus is constant, so that if the volume of the nucleus increases the cytoplasm volume will increase to, and the cell will be larger. Theoretically, triploid cells will have higher dimensions than diploid cells. Increased cell size * Corresponding author: Bencsik Ioan, , bencsik54@animalsci-tm.ro in polyploids applies to all tissues and cells of the body. Thus by measuring and comparing the cell size we can distinguish diploid individuals and triploid. Polyploidy determination can be achieved by karyotyping (visualization and counting of chromosomes), flow cytometry, cell size measurement by Coulter Counter Channellizer or blood smear, silver staining of nucleolar organizing regions (RON), isozymes analysis using markers [10, 11]. Karyotyping is the most accurate method, but the technique is slow and laborious [12,13]. Flow cytometry is quick method but the equipments are very expensive. Coulter Counter analysis is rapid, equipment is cheaper. Measurement of the size of blood cells in the smear method is quite accurate [10,11]. Analysis RON is the cheapest and easier to perform than karyotyping, it can be applied without sacrificing the young fish. The principle of the method is based on determining the number RON (1 RON per haploid genome) [14,15]. 113

2 Triploid fish (3n) are sterile, but diploid fish (2n) are not. Triploidisation is carried out in the early stages after fertilization by thermal shock, osmotic hyper pressure, electric shock, preventing removal of the second polar world. 2. Materials and methods 2.1 Blood smear preparation technique Blood smear is prepared following the next steps: blood sampling, spread the blood drop on the slide, fixation and staining. Blood sampling:- blood is collected by cardiac puncture, first drop is removed, the second drop is collected on clean slide. Afterwards the blood drop is spread by using another slide (spreader). The spreader is placed at a 45 angle and backing into the drop of blood. The spreader catches the drop and it spreads by capillary action along its edge, then by a quick and smooth motion blood is stretched from one to the other end of the slide. First fixation (physical fixation) was done immediately by shaking the slide in the air to avoid the deformation of the erythrocytes. Staining was done using the panoptical Pappenhein method. In this method May- Grünwald and concentrated Giemsa (4-10%) solutions were used. Staining was done by performing the following steps: - Slide with the blood smear was placed on a support in a Petri dishfrotiul, and May-Grünwald solution was poured; smear was left for 2-5 minutes in contact with May-Grünwald solution (chemical fixation) - To remove May-Grünwald the same number of bidistiled water were pored and left on for 1-3 minute. - The dye was removed without washing the slide and then the smear was covered with Giemsa solution. Blood smear was left in contact with Giemsa solution for minutes. During this step was strengthening the staining of acidophilic, neutrophile and basophile leukocyte components colored with May-Grünwald solution and the azurofile granulations in lymphocytes and monocytes. - In the end the Giemsa solution was removed and the smear was washed with water and then placed on a support and left there to be dryed on the air. The smear is correctly stained if the final color is pink or red-violet, if the color is blue or violet the smear was left for a too long period in contact with the dye. Microscopic examination of stained blood smears was made using immersion objective Triploidy induction Rainbow trout reproduction cycle starts when the water temperature reaches 8-10 C; those are the natural conditions which are necessary to induce this process and our experiments were performed under these conditions. Eggs were fertilized by using the wet method. Collected eggs from several females were placed in a vessel and then the semen (for eggs that are collected from one female is necessary the semen to be collected from 2 males). Afterwards, the eggs and the semen were mixed in the vessel. After homogenization, the vessel was placed in water for temperature equilibration, water that was used for semen activation. After 5 minutes from sperm activation and eggs fecundation, thermal shock was induced to retain the second polar body inside the fertilized egg and for triploidy generation. Thermal shock was realized in 28 C water. To induce the thermal shock we have used a thermostatically controlled water bath from stainless steel. Thermal shock was applied for 5-10 minutes. After that the eggs were counted and incubated. We have obtained and counted approximately eggs after the thermal treatment. Eggs from the control group after fecundation were put directly to incubation step. During the incubation period dead eggs, white colored, have been removed on a daily basis and counted through all 38 days incubation period. During the whole period of incubation until hatching 340 C were summed. For statistical interpretation of the obtained data and graphs preparation a special program was used. 3. Results and discussion To establish the triplody induction rate in rainbow trout embryos we started from the idea that thermal shock applied after 5-10 minutes after fertilization will block the elimination of the second polar body (2n + n = 3n). From the experimental group formed from triploid trout we have chosen randomly 30 specimens that were 114

3 slaughtered. After slaughtering the blood was collected and the smears prepared. Erythrocytes were examined on blood smears, on minimum 10 visual fields. To determine the exact dimensions for erythrocytes we measured both, cell area and cell perimeter. Results from these measurements done on triploid trout are presented in Table 1. From those data we observe that the erythrocytes area has an average of μm ² with values ranging between 32.7 to 52.8 μm². Erythrocytes perimeter average is μm, with minimum and maximum variation between μm. Figure 1. Eritrocytes from triploid trout Figure 2. Eritrocytes from diploid trout Table 1. Area and perimeter measurements results of erythrocytes from triploid rainbow trout Ref. no. Number of measurements Area (µm²) deviation Perimeter (µm) deviation , Average

4 Table 1 data were statistically reworked with a special PC program and the results are presented in Table 2. Table 2. The statistical interpretation on area and perimeter on tetraploid rainbow trout erythrocytes Ref. No. Specification x ±sx deviation Variance (s²) Coefficient of variation (C.V.) 1 Aria 42.44± Perimeter 25.40± From the Table 2 data can be observed that the area has an average and error of the mean value of ± 1.26 μm ², standard deviation has a value of 6.8, and coefficient of variation is 16.02; which means that our measurements have medium variability. In case of erythrocytes perimeter, media and medium error of the mean was ± 0.36 μm, standard deviation had a value of 1.9 and the coefficient of variation has a value of 7.4 which represents a small variation of this character. In case of control group the measurements for area and perimeter are of erythrocytes from diploid rainbow trout are presented in Table 3. We can observe that the average of the area measurements is 24.10µm² and the perimeter 19.30µm. Table 3. Results of area and perimeter of erythrocytes from diploid rainbow trout. Ref. no. Number of measurements Aria (µm²) deviation Perimeter (µm) deviation Average Tabel 4. The statistical interpretation on area and perimeter on diploid rainbow trout erythrocytes Ref. No. Specification x ±sx deviation Variance (s²) Coefficient of variation (C.V.) 1 Aria 24.10± Perimeter 19.30± From the Table 4 data can be observed that the area has an average and error of the mean value of ± 2.09 μm ², standard deviation has a value of 5.53 and coefficient of variation is 22.94, which means that our measurements have high variability. In case of erythrocytes perimeter, media and medium error of the mean was ± 0.94µm, standard deviation had a value of 2.5 and the coefficient of variation has a value of which represents a medium variation of this character. From the above presented data we can observe that the difference between area measurements in erythrocytes from triploid trout and the erythrocytes from diploid trout is 18.34µm²; which shows that erythrocytes from triploid individuals are having a higher area by 1.76 than diploid individuals. The difference observed on erythrocytes perimeter from triploid and diploid individuals is 6.1μm, which shows that erythrocytes from triploid trout are having a higher perimeter by 1.31 than diploid individuals. 116

5 4. Conclusions Erythrocytes area measured on triploid and diplod individuals can represent an indicator in establishing the polyploidy grade. Erythrocytes perimeter measured on triploid and diplod individuals can represent an indicator in establishing the polyploidy grade. Erythrocytes area at triploid individuals, measured in µm², is 1.76 bigger than in diploid trout. Erythrocytes perimeter at triploid individuals, measured in µm, is 1.31 bigger than in diploid trout Coefficient of variation in area measurement case is medium; this means that this measurement method has an acceptable precision and accuracy to be used in determination of the poliploidy grade in rainbow trout. Coefficient of variation in perimeter measurement case is low; this means that this measurement method has an acceptable precision and accuracy to be used in determination of the poliploidy grade in rainbow trout. References 1. Peruzzi, S., Chatain, B. Pressure and cold shock induction of meiotic gynogenesis and triploidy. Aquaculture, 2000, 189: Sellars M.J., Coman F.E., Degnan B.M., Preston N.P., The effectiveness of heat, cold and 6- dimetilaminopurine shoks for inducing tetraploidy in the kuruma shrimp, Marsupanaeus Japonicus (Bate), Journal of Shellfish Research 2006, 25 (2), Beaumont, A., Hoare, K., Biotehnology and genetics in fisheries and aquaculture, Blakwell Publishing, Bohl, M., Zucht und Production von Suβwasserfischen, Verglas Union Agrar, 1999, p Dunham, R.A., Aquaculture and fisheries biotehnologie, Genetic approaches, CABI Publishing, Cambridge, USA, Haffray, P. et al., Gonad development, growth, survival and quality traits in triploids of the protandrous hermaphrodite gilthead seabream, Sparus aurata (L.). Aquaculture, 2005, 247: Kalbassi, M., Johari, S.A., A study on the production possibility of all-female triploid rainbow trout (Oncorhznchus mikiss), J.Sc.& Technol.Agric. &Natur.Resour., 2008, 12, Kapuscinski A., R., Miller L., M., Genetic guidelines for fisheries management, University of Minesota, 2007, p Quillet, E., Foisil, L., Chevassus, B., Chourrout, D., Liu, F.G., Liu, F.,G., Production of all-triploid and allfemale brown trout for aquaculture, Aquat.living Resour., 1991, 4, Benfey, T.J., Sutterlin, A.M. and Thompson, R.J., Use of erythrocyte measurements to identify triploid salmonids, Canadian Journal of Fisheries and Aquatic Sciences, 1984, 41, Garcia-Abiado, M.A.R., Dabrowski, K., Christensen, J.E., Czesny, S. and Bajer, P., Use of erythrocyte measurements to identify triploid saugeyes. North American Journal of Aquaculture, 1999, 61, Colihueque, N., Iturra, P., Diaz, N., Veloso, A., Estay, F., Karyological analysis and identification of heterochromosomes in experimental gynogenetic offspring of rainbow trout (Oncorhynchus mykiss, Walbaum) Rev.Brasil.Genet, 1992, 15, 3, Quillet, E., Aubard, G., Queau, I., Mutattion in a sex-determining gene in rainbow trout Detection and genetic analysis, The American Genetic Association, 2002, 93: Phillips, R.B., Zajicek, K.D., Ihssen, P.E. and Johnson, O., Application of silver staining to the identification of triploid fish cells., Aquaculture, 1986, 54, Strunjak-Petrovic,I., Coz-Rakovac, R., Topic Popovic, N., Micronucleus occurrence in diploid and triploid rainbow trout (Oncorhynchus mykiss Walbaun), Vet. Med. - Czech, 2003, 48, (8),