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Cytogenetic variations in Clarias species 279caped plastic test tube. The content wascentrifuged at 2,700 rpm for 3 minutesusing micro angle centrifuge.2. Hank’s balance salt solution plusphytohaemagglutinin separation: 1ml of HBSS and 0.2 ml of 1 mg ml -1 PHAwere added to 1 ml of anticoagulatedblood. The contents were mixed andcentrifuged at 2, 700 rpm for 3 minutes toensure proper settling out of erythrocytes.3. Phytohaemagglutinin separation: 1ml of anticoagulated blood was dilutedwith 0.2 ml of 1 mg ml -1 PHA. This wasmixed by gentle inversion in a sterile 5 mlcapped plastic test tube. The content wascentrifuged at 2,700 rpm for 3 minutes.In each case, the leucocyte richsupernatant was used as inoculum.Blood Leucoyte Culture Techniques: Thecomplete culture medium used composed of 20 mlof HBSS, 2 ml of Eargle’s Essential CultureMedium. Laboratory prepared phytohaemagglutininwas added to make up either 5 % of theentire culture medium. Furthermore, freshlyprepared rabbit sera were added to make up 20 %of the entire culture medium. The completemedium was aseptically filtered using membranefilter. The complete culture medium (5 ml) wasplaced in sterile tube. Various glass and plasticsterile test tubes were tried for culturing of fishleuocytes. About 0.05 ml of leucocyte richsupernatant was aseptically placed on the bottomof the culture tube containing 5 ml of the completeculture medium using a sterile 1 ml syringe fittedwith 26-gauge 1¼-inch needle. Culture vials wereincubated at 36 ± 1 0 C for 72 hours.Chromosome Harvesting and SlidePreparation Techniques: The cultured leucocytecells were arrested 3 – 6 hours with 0.3 µg ml -1 ofcolchicine solution before the end of the 72 hoursculture period to give good mitotic chromosomespread for karyotyping (Eyo, 1997). At the end ofincubation and metaphase arresting period, thecells were harvested by centrifuging at 1000 rpmfor 5 minutes. The supernatants were discardedand the white button-like residue given 5 ml of0.075 M KCl hypotonic treatment for 10 minutesand expirated using a micropipette. The residueswere obtained through centrifugation and thesupernatants discarded. 2 ml of freshly preparedcold Carnony’s fixative (absolute ethanol andglacial acetic acid (3:1)) was added to the residuecells, expirated to disperse the cell pellets and leftstanding for 15 minutes. The fixation process wasrepeated twice at 15 minutes intervals. After thefinal centrifugation, the fixative was decantedleaving about 3 drops in which to re-suspend thecells through expiration. The chromosome spreadwas accomplished using a modification of Mellman(1965) air-dried technique.Thoroughly clean grease free slides(commercially pre-cleaned slides) were dipped intodeionized water and frozen. The chilled slideswere observed for cleanliness by adherence ofwater film. Excess fluid was shaken off and onedrop of cells suspension was placed on the centerof the wet slide inclined at an angle of 45 0 . Ablotter placed underneath the slides removedexcess fluid. Drying was completed immediatelyby blowing the slides to promote evaporation andby gentle warming under 80 watts electric bulb.The quality of slides was checked using ahand lens. Inadequate spreading was correctedby re-fixation of the cells. Slides were stained in10 % buffered Giemsa stain for 5 minutes, anddehydrated for 1 min each in two jars of acetone,one jar of acetone and xylene solution (1:1) andfinally one jar of xylene. Slides were observed todestain in acetone when shaken and or agitated.Thus, shaking and agitation was avoided. Slideswere scanned for metaphase chromosomes usinga binocular light microscope at X 1500magnification. Slides with good metaphasechromosomes were processed into permanentmounts. The chromosomal arm lengths weremeasured to the nearest micron using ocularmicrometer.Chromosome Analysis Nomenclature: Tenslides per species showing good metaphasechromosome were analysed. The frequency ofdiploid chromosomes number per species wasrecorded. The maximum or minimum mean andmodal chromosome number were computed foreach species. F-LSD was employed to separatethe differences in modes. The arm ratio r, (longarm L/ short arm S) and the centromeric index i(100 x short arm/total length of chromosome)were calculated. Furthermore, each chromosomewas classified into any of the six groups; M, m,sm, st, t, and T based on Levan et al. (1964)classification (Table 1).Table 1: Chromosome arm nomenclaturerelevant to Clariid cytotaxonomyTerm Location d value r valueM Median point M 00 1.0m median region m 0.0 – 2.5 1.01 -1.7Sm Submedian region Sm 2.5-5.0 1.7-3.0St subterminal region st 5.0-7.5 3.0-7.0t(a) terminal region t 7.5-10 7.0-00T(a) Terminal point T 10-0 00
EYO, Joseph Effiong 280Based on the nomenclature, the karyotypicdifference within and between the Clarias specieswere ascertained.length percentage distribution of 8.80 % to 4.40% with bimodal chromosome percentage totallength of 6.60 %, made up of 40.00 % medianRESULTSCytogenetic Variations among four ClariasSpecies from Anambra River, Nigeria: Tables2 and 3 illustrates the modal chromosomes andkaryotype morphology distribution respectivelyamong the Clarias species of Anambra river. Themodal metaphase chromosome numbers were 2n= 48 in C. ebriensis, 2n = 48 in C. albopunctatus,2n = 56 in C. gariepinus and 2n = 56 in C.anguillaris. They were characterized by a highproportion of meta-submetacentric chromosomesand low proportion of acrocentric chromosomes.All female karyotypes exhibited a heteromorphicpair suspected to be sex chromosome complex.The following formulae were established for themale clariids; C. ebriensis 6m + 22sm + 20a FN=76; C. albopunctatus 4m ± 22sm + 22a FN = 74;C. gariepinus 8m + 24a FN = 88; and C.anguillaris 8m + 26m + 22a FN = 90. The femalekaryotype morphologies were C. ebriensis 6m +23sm + 19a FN = 77, C . albopunctatus 4m ±23sm + 21a FN = 75, C. gariepinus 8m + 25sm +23a FN = 89 and C. anguillaris 8m + 27sm + 21aFN = 91. Furthermore, figures 1 – 4 displaysrepresentative metaphase chromosomes of malesand females Clarias species from Anambra River,Nigeria.The karyotype distributions and idiogramswere developed based on chromosomenomenclature of Clarias species using theircentromeric indices and presented on figures 5and 6, 7 and 8, 9 and 10 and 11 and 12 for malesC. ebriensis, C. albopunctatus, C. gariepinus andC. anguillaris respectively. In C. ebriensis, six (6)chromosomal length distributions occurred withmodal chromosome percentage total length of7.00 %. Seventy five percent of the modalchromosomes were submedian chromosomeswhile 12.50 % were both median and terminalchromosomes (Figure 5 and 6).In C. albopunctatus, the chromosometotal length percentage distributions exhibitingbimodal length peaks of 9.01 % made up of 50.00% submedian and 50.00 % terminalchromosomes; and 7.50 % made up of 100%submedian chromosomes (Figures 7 and 8). C.gariepinus exhibited chromosome total lengthpercentage distributions of 8.37 % and 3.90 %with modal chromosome percentage length of 5.58%. Fifty-seven percentage (57.00 %) of the modalchromosome percentage total length distributionwere submedian chromosomes while 29.00 %were terminal chromosomes with only one (14.00%) median chromosome (Figure 9 and 10).Similarly, C. anguillaris exhibits chromosome totalMaleFemaleFigure 1: Metaphase chromosomes ofClarias ebriensis from Anambra river,Nigeria (bar = 10µ)MaleFemaleFigure 2: Metaphase chromosomes ofClarias albopunctatus from Anambrariver, Nigeria (bar = 10µ)MaleMaleFemaleFigure 3: Metaphase chromosomes ofClarias gariepinus from Anambra river,Nigeria (bar = 10µ)FemaleFigure 4: Metaphase chromosomes ofClarias anguillaris from Anambra river,Nigeria (bar = 10µ)chromosomes and 60.00 % terminalchromosomes; and 5.52 %, made up of 100 %submedian chromosomes. The chromosome totallength has been categorized into 13 length groups(Figures 11 and 12).
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Animal ResearchInternational ®Anim
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Influence of dietary protein conten
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MGBENKA, Bernard Obialo et al. 216T
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EKWUNIFE, Chinyelu Angela 220repeat
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EKWUNIFE, Chinyelu Angela 222Table
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Animal Research International (2005
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Page 77 and 78: EYO, Joseph Effiong 286Cyprinidae,
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