Thermal induction of tetraploidy and assessment of selected DNA quantification methods in African sharptooth catfish, Clarias gariepinus
Date
2022-04
Authors
Journal Title
Journal ISSN
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Publisher
Stellenbosch : Stellenbosch University
Abstract
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.
AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar.
Description
Thesis (MScAgric)--Stellenbosch University, 2022.
Keywords
Sharptooth catfish -- Breeding, Tetraploidy, DNA, Clarias gariepinus -- Africa, Nucleolar organising regions, UCTD