Thermal induction of tetraploidy and assessment of selected DNA quantification methods in African sharptooth catfish, Clarias gariepinus

Senyolo, Thalepo (2022-04)

Thesis (MScAgric)--Stellenbosch University, 2022.


ENGLISH ABSTRACT: Producing viable African sharptooth catfish (C. gariepinus) tetraploids has become imperative and potentially beneficial in aquaculture for mass production of sterile triploids, which can be generated by mating natural diploids with tetraploids. The objectives of the current study were to assess the correct shock initiation time between egg fertilisation and heat shock induction of tetraploidy and to validate three methods for DNA quantification namely flow cytometry (Sysmex® PI method), chromosome preparations (Giemsa staining) and silver nitrate staining to quantify Nucleolar organising regions (NORs). Properly conditioned broodstock with good physical condition (CF 3.1 ± 0.3%), pre- fertilised egg quality (> 1000μ egg diameter) and fecundity (RSI >11%) were employed for spawning, since low quality fertilised eggs (< 1000 μ egg diameter) could not survive ploidy manipulation procedures. To successfully induce tetraploidy, fertilised eggs incubated at 25 °C ± 0.1 were immersed in a water bath (40.5 °C ± 0.1) for 2 minutes at shock initiation times (IT) from 40 – 50 minutes post fertilisation. The IT treatments within this range were spaced one minute apart i.e. 40 – 45 minutes and the remaining IT treatments were 30 seconds apart i.e. 45.5 – 50 minutes. Fertilisation completion was set at 15 seconds after the addition of activated sperm to eggs. The thermal shock led to a decrease in hatching and larval survival percentage, and with a progressive trend from about 41% (40 minutes post-fertilisation treatment) to 1% (50 minutes post fertilisation treatment). Ploidy status determination of African sharptooth catfish was evaluated by testing procedures respectively developed for chromosome preparations, silver nitrate-NOR staining and flow cytometry. Flow cytometry was selected as the preferred DNA quantification method when excluding erythrocyte karyotyping (thrombosis issues), since the other procedures proved to be inconsistent, tedious and unreliable. The slightly modified CyStain® PI Absolute P protocol used for flow cytometry analysis in the current investigation proved to be a rapid and efficient method for processing and determining the karyotype of minced 2 DPH larvae or finclips (non-destructive option). Production of tetraploid offspring (60 – 90+%) was predominant when heat shocked from the 45 minute to 47 minute setpoint initiation times post-fertilisation, with peak frequency of occurrence (>90%) at 47 th minutes post fertilisation induction setpoint. Microscopic determined nuclear sizing (erythrocytes) and nuclear sizing electronically by Coulter counter TM efficacies should be tested for comparison in future studies to flow cytometry DNA quantification in terms of ploidy verification accuracy, test rapidity, and cost effectiveness. The outcome of the current work unlocks the opportunity to produce all triploid C. gariepinus offspring by crossing female tetraploid with male diploids, and could potentially facilitate in the future commercial production of triploid African sharptooth catfish in regulated eco- sensitive regions.

AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar.

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