Cryopreservation of African Catfish: comparison of

embryo viability between blastula stages and

gastrula stages after vitrification
Garini, D. Aa; Nugroho, R. Pa; Mukti, A. Tb; Mubarak, A. Sc; Luqman, E. Md

a
Aquaculture of Fisheries and Marine, Airlangga University, Mulyorejo 60115
Surabaya, East Java, Indonesia
b
Department of Fish Health and Aquaculture Management, Fisheries and Marine,
Airlangga University, Mulyorejo 60115 Surabaya, East Java, Indonesia
c
Department of Marine Affairs, Fisheries and Marine, Airlangga University, Mulyorejo
60115 Surabaya, East Java, Indonesia
d
Department of Veterinary Anatomy, Veterinary Medicine, Airlangga University,
Mulyorejo 60115 Surabaya, East Java, Indonesia

ABSTRACT
Corresponding author: Akhmad Taufiq Mukti. Department of Fish Health and
Aquaculture Management, Fisheries and Marine, Airlangga University, Mulyorejo
60115 Surabaya, East Java, Indonesia. Akhmad Taufiq Mukti atm.mlg@gmail.com

1. INTRODUCTION

Fish farming technology is increasingly developing from traditional fish farming to

intensive and supra-intensive fish farming. The rapid development of cultivation
technology is of course accompanied by various constraints such as lack of seed supply
due to limited spawning season which greatly affects the success of cultivation business.
However, there have been many technological advances to meet the constraints of
cultivation in terms of quality and quantity through storage techniques for gamete cells
(sperm and oocytes) to embryos using cryopreservation technology. In this study, the
object of genetic material to be cryopreserved is the embryo of African Catfish (Clarias
gariepinus). African Catfish is a freshwater cultivated fish produced by hybridization or
cross-breeding which often has the possibility of experiencing a decline in genetic
quality due to uncontrolled spawning (inbreeding). To improve the quality and quantity
of catfish seeds, it is appropriate to conduct controlled breeding of superior sires and
storage of embryos using the freezing method in order to obtain superior seeds of the
same quality outside the continuous spawning season.

Embryo cryopreservation in fish is an efficient reproductive technology and helps

increase the effectiveness of cultivator production [1]. One of the successes of embryo
cryopreservation technology is by freezing it at the right phase. The use of the blastula
phase in this study because it has a chorion layer which has high permeability properties
so that it can be easily penetrated by cryoprotectants and can increase the success of the
cryopreservation process [2]. The blastula stage is characterized by repeated divisions of
the fertilized egg. Blastula cells form an epithelial layer (cover), called the blastoderm,
covering the fluid-filled cavity (blastocoel) and on the outside, there is an epiblast [3].
This study also uses the post-gastrula phase which has a high probability of supporting
cryopreservation success, because in the post-gastrula phase the chorionic layer of the
embryo begins to thicken which will be more effective in protecting genetic material
from temperature shock (cold shock). The post-gastrula stage is characterized in that it
is followed by organogenesis when individual organs develop within the newly formed
germ layers. Each layer gives rise to certain tissues and organs in the developing
embryo [4]. Embryo cryopreservation techniques in the blastula phase were developed
in several fish including Zebrafish [2], Rainbow Trout [3], Chum Salmon [4], and
Medaka [5] while in the post gastrula phase it was developed using Tambaque fish
embryos [6].

Based on the description above, the aim of the current study is to conduct research on
the differences in the use of the embryo phase at the blastula and gastrula stages for a
cryopreservation study model using the vitrification method as a comparison to support
the success of embryo cryopreservation, especially for African Catfish (Clarias
gariepinus).

2. MATERIAL AND METHODS

2.1 Embryos Production - The object of research in this study was African Catfish
(Clarias gariepinus) by induced breeding using GnRH hormonal induction
(Ovaprim Syndel®). The dose of GnRH hormonal induction used is 0.5 mL/kg
of fish weight. The broodstock to be spawned is 2 years old and weighs 1.4 - 1.5
kg for the female and 1.1 - 1.2 grams for the male. The broodstock of African
Catfish is injected once with a span of time for 6-12 hours. Induced breeding
process is done by mixing eggs and sperm that had been diluted with NaCl
physiological 0.9% 1: 100 ratio.
After fertilization, the embryos are placed in a hatching pond measuring
40x20x30 cm which has been equipped with a thermometer, heater and aeration.
Observation of the stages of embryo development was carried out under a light
microscope Olympys Cx41 with 100x magnification to the blastula and post-
gastrula phases. Observation of embryogenesis to blastula stage takes about 4
hours from the initial duration of the fertilized egg while observing
embryogenesis to post gastrula stage takes about 8 hours from the initial
duration of the fertilized egg.
2.2 Ekuilibration - Before the cryopreservation process was carried out, embryos that
had reached the blastula and post-gastrula embryonic stage were equilibrated
first with a cryoprotectant solution in a petri dish, then added the extender
solution into the cryotube after the equilibration process. The equilibration
process with cryoprotectant was carried out at 27 ° C with immersion time for 15
minutes with treatments consisting of treatment 1; 10% PROH, treatment 2; 20%
PROH, treatment 3; 30% PROH, treatment 4; 10% EG, treatment 5; 20% EG
and treatment 6; 30% EG. Comparators for all treatments used fresh embryo
specimens as control treatment. After equilibration with cryoprotectants, 30
embryos were put into a 2 mL cryotube with the addition of a 1 M sucrose
extender solution to each of the 1.8 mL cryotubes. The equilibration process
with the extender is carried out as quickly as possible with an equilibration time
of 15 minutes before being put into a liquid nitrogen container.

2.3 Vitrification - Samples from 30 embryos of blastula and post-gastrula embryo

stages that had been treated were stored by the vitrification method in liquid
nitrogen at -196°C. The embryos are stored at intervals of 24 hours.

2.4 Thawing - After the embryo storage process is carried out, the next step is the
thawing process which is the cryotube liquefaction process. The thawing process
aims to reduce the temperature or adapt the embryos on the cryotube. Thawing
was carried out with temperature fluctuations using a freezer at -18 ° C for 15
minutes and a waterbath at 27 ° C for 15 minutes. After thawing, the embryos in
cryotube containing sucrose were drained, then transferred to a petri dish
containing 0.9% physiological NaCl for 5 minutes.

2.5 Assessment of Embryos Viability -

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