REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE.

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REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED
AQUACULTURE
Name of the Student
Name of the University
Author Note

1REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE
Abstract
European eel (Anguilla anguilla) is now critically endangered from European
continent and there is no satisfactory sign of recovery can be seen for last 25 years. The
livestock is declining day by day. European Union has taken the Council regulation
1100/2007 to establish some measures for recovering critical livestock of that fish. Human
fishing and consumption of Eel has been amplified as a result these fishes are not having the
scope to re-establish themselves in nature with stability1. EU has taken an initiative to
control the decreasing livestock of eel by launching PRO-EEL project, which will help in the
process of conserving the eggs and gametes and produce better quality hormonal control in
the fish2. Self-sustained aquaculture is one of the most acknowledged marine fish culture
acquired by the expert and professional. However, the process can be used to conserve
critically endangered species and to improve the livestock of marine or aquatic organisms.
This research to analyse the effectiveness of self-sustained aquaculture on crucial livestock
status of European Eel fishes with the reference of water salinity impact on the egg size and
fertilization. The migratory nature of the eels are important for their lifecycle but the salinity
with its associated factors are changing day by day. Constant changes in the sea water is
affecting the fertilization rate of European eels. The research or project is to observe whether
the salinity is important for reproduction of eels and how the salinity gradient acts on the
fertilization rate. In the research it will be observed that activation salinities between 30–40
practical salinity unit (psu) produced higher quality eggs and generally larger egg diameters.
The chorion diameter is reached in its maximal value which is 1642 ± 8 μm at 35 psu. The
1 Stein, F. M., Wong, J. C., Sheng, V., Law, C. S., Schröder, B., and Baker, D. M. (2016). Erratum to:
First genetic evidence of illegal trade in endangered European eel (Anguilla anguilla) from Europe to
Asia. Conservation Genetics Resources, 4(8), 539-539.
2 da Silva, Filipa FG, et al. "Interactive effects of dietary composition and hormonal treatment on
reproductive development of cultured female European eel, Anguilla anguilla." Animal reproduction
science 171 (2016): 17-26.

2REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE
research will show some positive relationship between the salinity and the buoyancy.
Different types of eggs and their different attributes such as chorion diameter, perivitelline
space, yolk diameter has been studied in the research. The study aims to relate the ionic
composition of water with the fertillization rate and conditions of eggs of European Eels,
which are needed to be conserved.
Introduction
Eel has high socio-economic importance in Europe and regular degradation is one of
the major concerns over the continent. The migratory nature of eel is important for its life
cycle and due this varied reproductive nature, researchers are highly interested towards these
organisms. Eel has two major developmental or growth stages such as continental and
oceanic3. Eels are migrated to spawn in the Sargasso Sea and larvae re found in the form of
yolk sac in the eggs. Larvae are fed and transformed into the leaf-shaped larvae, which are
also called leptocephali. These leptocephali larvae are flowed in the North American Sea and
the European Coast in 1 to 2 years of timespan due to Gulf Stream and North Atlantic
Stream. In the coastal areas, the eels are transformed into glass eels. The large portion of the
glass eels remains in the coastal and estuarine regions. The glass eels developed and
pigmented; as a result, yellow eels are found. After some times, yellow eels are transformed
into yellow-green eels. Eels are developed into the matured phase during 6-16 years and
found in the cold northern areas. 4. Head of the eels transformed into the silver eels. Finally,
3 Arai, T., SR Abdul Kadir, and N. Chino. "Year-round spawning by a tropical catadromous eel
Anguilla bicolor bicolor." Marine Biology 163.2 (2016): 37.
4 Moravec, František, and Tomáš Scholz. "Macroparasites and their communities of the European eel
Anguilla anguilla (Linnaeus) in the Czech Republic." Folia parasitologica 62 (2015): 033.

3REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE
the silver eels are migrated to the downstream and development of ovaries in females and
testes in male eels are disrupted in this phase5. Different maturation phases are identified in
the life cycle of eel fish and this complex life cycle is one of the most relevant causes
considered for abundance of its livestock.
Vulnerability of eel fishes are responsible for its single time reproduction in the entire
lifecycle. The bottleneck effect is high on this species and the hatchery and marine
technologies are trying to control the brood stock of eel fish. Yellow and Green eels are
affected mostly by human fishing and other environmental causes as their habitat are
destroyed or quality of water in sea is deteriorated. Fisheries, habitat quality, parasites and
diseases, pollution for infection and contaminations are some important anthropological and
environmental factors, which are affecting European Eel brood stock. According to ICES
(The International Council for Exploitation of Sea), lack of human awareness and morality
leads declining of eel livestock. Human fisheries are responsible for mortality of both
juvenile and mature ones 6 .Continuous live fishing is the key factor and strong indication for
the reducing stock of eel. Rate of fish production of European eel is less than Japanese Eel
fishes and by analysis conditions of prolarvae and gametocytes helps to evaluate quality of
eggs 7. Crisis of habitat is another reason behind the eel extinction as sustainability is reduced
in both quality and in terms of the number. Regulation of rivers is the cause of habitat crisis
for Eel and make a barrier in migration of eel fishes. Obstacles in the migration through
5 Geffroy, Benjamin, and Agnès Bardonnet. "Sex differentiation and sex determination in eels:
consequences for management." Fish and fisheries 17.2 (2016): 375-398.
6 Bevacqua et al., "A demographic model for the conservation and management of the European eel: an
application to a Mediterranean coastal lagoon." ICES Journal of Marine Science (2019).
7 Itakura, Hikaru, et al. "Feeding, condition, and abundance of Japanese eels from natural and revetment
habitats in the Tone River, Japan." Environmental biology of fishes 98.8 (2015): 1871-1888.

4REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE
downstream is responsible for high rate of mortality in eel fishes during their migratory
phases. Anguillicoloides crassus is one of the nematode parasites which is located in the gills
of eel fishes. Heavy distribution of the parasites among the European continent is predicted
as one of the main causes for poor spawning and disruption of migration in silver eels due to
health issues8. In recent decades the biological livestock of European eel fishes are declined
at the rate of 90% and the population size is decreased for environmental and the ecological
effect of their genetical bottleneck effect 9. This effect is unfavourable for allele conservation
among European eels as the effective and progressive alleles are flowed to another direction
and cannot exist alone due to demographic differences.
Method and Result
Eels were transported to the university from the freshwater into a tank of recirculation
system at a density less than 30kg m3. The water is adjusted by adding Tropic Marine Sea
Salt to adjust the salinity of the water to approximately 35psu and the temperature is attuned
at 20˚C under dim light, which has similarity with the natural day light10. The brood stock
was not provided any feedings to prevent ceasing of food intake in eels. The female broods
were injected with the Salmon Pituitary Extract. Within 24 hours of injecting the SPE,
8 Pegg, Josephine, et al. "Head morphology and piscivory of European eels, Anguilla anguilla, predict
their probability of infection by the invasive parasitic nematode Anguillicoloides crassus." Freshwater
biology 60.10 (2015): 1977-1987.
9 Neves, João Bernardo, Filipe Martinho, and Miguel Ângelo Pardal. "Effect of illegal glass eel
(Anguilla anguilla) fishery on estuarine fish stocks: a case study in the Mondego Estuary, Portugal." Marine and
Freshwater Research 69.11 (2018): 1692-1703.
10 Asturiano, J. F., et al. "First production of larvae using cryopreserved sperm: effects of preservation
temperature and cryopreservation on European eel sperm fertilization capacity." Reproduction in Domestic
Animals 51.4 (2016): 485-491.

5REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE
maturation inducing steroid is added to the female broods. Five female fishes are taken and
fertilized with sperm of another 3-4 male eel fishes. The salinity was adjusted by using
electronic conductivity meter. The pH of the water was measured 6.5-10.0 by the pH
indicative strips and the gamete samples were mixed and distributed for different treatment
with different psu such as 30,35,40,45 and 50 psu 11. After mixing the gametes the replicates
were immediately activated by injecting 11.7ml of activation medium using syringe. Female
eels were labelled as f1,f2,f3,f4,f5 and let them free in the tank for spawning of egg hatching.
11 Sørensen, Sune Riis, et al. "Effects of salinity and sea salt type on egg activation, fertilization,
buoyancy and early embryology of European eel, Anguilla anguilla." Zygote 24.1 (2016): 121-138.

6REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE
Discussion
No relation was
found in between the female body and characteristics of eggs. The average diameter for egg
chorion was from 1207 ± 212 to 1338 ± 203 μm in the experiment. In the research , it has
been observed that average yolk diameter for different eel females after rdifferent. The yolk
Figure :1
(Source:Static.cambridge.org, 2019)
Figure :2
(Source: Static.cambridge.org, 2019)
Figure :3
(Source: Static.cambridge.org, 2019)

7REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE
diameters are ranged from 1207 ± 212 to 1338 ± 203 μm in the salinity gradient. For the
experiment the mixed model of ANOVA test shows variance component are not much
affected in different salinity. The relation between female eels and the salinity is variant in
nature and the model variance of the experiment is expanded to 99.7%. However, female
varienbce is not expanded in that range and it is limited to only at 0.28%. the yolk diameter of
female eels are constant in various salinity range and mixed model for the experiment shows
no effect of salinity on the diameter of egg yolk.
It can be said that no significant difference can be found among the female body and
percentage of egg fertilization. Across the salinity gradient the success ranged from
22.1±18.6 to 60.9 ± 16.9% for egg fertilization. If every salinity range is considered, it can be
interpreted that the 30-40 psu is most effective salinity treatment for fertilization. The salinity
gradient has shown the most effectiveness for the random model effect. Initially there is such
relation between the salinity and the cleaved fertilised eggs as salinity is ineffective on the
cleavage formation of eggs. It has been observe in the research that the overall rate of regular
cleaved fertilized eggs , ranged from 0.65 ± 0.98 to 14.71 ± 21.57%. Among the female
effects, the result of f2 and f4 females are most significant and the regular cleaved
fertilization rate is declined across the salinity gradient. In the research the fertilized eggs
showed mostly regular cell cleavages. It has beeb observed in the research that salinity had an
impact, where the percentage of fertilized eggs declined at the 50 psu treatment. In the
experiment no significance was found as no functionality of water salinity was observed over
egg chorion diameter. Female × salinity was enough significant according to the model’s
random effect as the VC (various component) was variance and that is equal to 99.7%. The
chorion was the largest for f1 and f2 in 30 and 35psu , f3 in 40-45 psu , f4 in 35-50 psu and

8REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE
fro f5 fishes psu is the same as f412. Figure 1 is representation of the relationship of salinity
with the egg size where egg size is dependent and salinity is an independent variable. The
result was noted after 3-5 hours of in vitro fertilization and include records regarding the yolk
diameter in fertilized eggs part A of the graph represents average egg size whereas part B-F
represent egg size of sample female from f1-f5. Figure 2 is the representation of the
percentage of fertilized eggs and sanity of the water with the reference of success in
fertilization rate and pattern of cleavage in the incubation salinity. Incubation temperature is
considered 20 ± 0.5˚C and the there are some sub categories which are assessed by the time
after fertilization and the incubation temperature. The parts for m A- F are similar in the
figure 2 as figure 1. In the Figure3. Box plots of egg chorion and yolk diameter after 3.5
hours of the post fertilization in part A. Part B of Figure :3 contains three images of (1)
Perivitelline space and a disk, (2) image of Egg with reducing PVS and (3)egg with
parthenogenetic cleavage. From figure 3 it can be said that female × salinity intersection test
was significant with the VC=23.1% as the success rate of fertilization is high in the every
labelled individual female eels except the f513. It has been observed that the lowest
fertilization rate was detected in 50 psu of water salinity. 58.7% model variance was
significant for the female eel fishes and 35% of the variance was significant for the
intersection experiment. T-1a egg category showed most common cleavage but salinity had
impact on the fertilized eggs in the sample water with salinity 50psu treatment. Salinity has
12 Politis, Sebastian N., et al. "Salinity reduction benefits European eel larvae: Insights at the
morphological and molecular level." PloS one 13.6 (2018): e0198294.
13 Di Biase, Andrea, et al. "Co-treatment with androgens during artificial induction of maturation in
female eel, Anguilla anguilla: Effects on egg production and early development." Aquaculture 479 (2017): 508-
515.

9REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE
significant impact on the distribution of fertilized eggs of T-1ab category but not in the f3
female eels.
The result shows that salinity has impact in the fertilization of eel eggs but for
different salinity unit. Salinity has effect on the fertilized eggs and very less effect on chorion
of eggs. Buoyancies are dependent on salinity for activation of fertilization of eggs and
incubation of samples14. The sinking speed of the eggs are recorded in the period of 210
minute from 7- 10.5 minutes. So it can be said that the buoyancy has been increased with the
gradient of salinity.
Conclusion
Inappropriate stimulation and enlargement of in vitro produced eggs of European eel,
(Anguilla anguilla), has been revealed to affect embryonic growth and producing. We
examined this occurrence by inspecting the impacts of salinity and various sea salt type on
egg dimensions, cell cleavage patterns and egg buoyancy. Egg diameter after activation,
using natural seawater adjusted to different salinities, varied among female eels, but no
consistent pattern emerged. The relationship of buoyancy of fertilised eggs are different with
the salinity gradient. The variation of the salinity for different salt can be found by the
execution of the research. The research has not come out with highly significant outcome but
the future researches will be helpful with the study.
14 Politis, Sebastian N., et al. "Salinity reduction benefits European eel larvae: Insights at the
morphological and molecular level." PloS one 13.6 (2018): e0198294.

10REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE

11REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE
Reference list
Arai, T., SR Abdul Kadir, and N. Chino. "Year-round spawning by a tropical catadromous
eel Anguilla bicolor bicolor." Marine Biology 163.2 (2016): 37.
Asturiano, J. F., et al. "First production of larvae using cryopreserved sperm: effects of
preservation temperature and cryopreservation on European eel sperm fertilization
capacity." Reproduction in Domestic Animals 51.4 (2016): 485-491.
Bevacqua, Daniele, et al. "A demographic model for the conservation and management of the
European eel: an application to a Mediterranean coastal lagoon." ICES Journal of Marine
Science (2019).
da Silva, Filipa FG, et al. "Interactive effects of dietary composition and hormonal treatment
on reproductive development of cultured female European eel, Anguilla anguilla." Animal
reproduction science 171 (2016): 17-26.
Di Biase, Andrea, et al. "Co-treatment with androgens during artificial induction of
maturation in female eel, Anguilla anguilla: Effects on egg production and early
development." Aquaculture 479 (2017): 508-515.
Geffroy, Benjamin, and Agnès Bardonnet. "Sex differentiation and sex determination
in eels: consequences for management." Fish and fisheries 17.2 (2016): 375-398.
Itakura, Hikaru, et al. "Feeding, condition, and abundance of Japanese eels from natural and
revetment habitats in the Tone River, Japan." Environmental biology of fishes 98.8 (2015):
1871-1888.

12REPRODUCTION OF EUROPEAN EEL TOWARD SELF-SUSTAINED AQUACULTURE
Moravec, František, and Tomáš Scholz. "Macroparasites and their communities of the
European eel Anguilla anguilla (Linnaeus) in the Czech Republic." Folia parasitologica 62
(2015): 033.
Neves, João Bernardo, Filipe Martinho, and Miguel Ângelo Pardal. "Effect of illegal glass eel
(Anguilla anguilla) fishery on estuarine fish stocks: a case study in the Mondego Estuary,
Portugal." Marine and Freshwater Research 69.11 (2018): 1692-1703.
Pegg, Josephine, et al. "Head morphology and piscivory of European eels, Anguilla anguilla,
predict their probability of infection by the invasive parasitic nematode Anguillicoloides
crassus." Freshwater biology 60.10 (2015): 1977-1987.
Politis, Sebastian N., et al. "Salinity reduction benefits European eel larvae: Insights at the
morphological and molecular level." PloS one 13.6 (2018): e0198294.
Politis, Sebastian N., et al. "Salinity reduction benefits European eel larvae: Insights at the
morphological and molecular level." PloS one 13.6 (2018): e0198294.
Sørensen, Sune Riis, et al. "Effects of salinity and sea salt type on egg activation,
fertilization, buoyancy and early embryology of European eel, Anguilla
anguilla." Zygote 24.1 (2016): 121-138.
Stein, F. M., Wong, J. C., Sheng, V., Law, C. S., Schröder, B., & Baker, D. M. (2016).
Erratum to: First genetic evidence of illegal trade in endangered European eel (Anguilla
anguilla) from Europe to Asia. Conservation Genetics Resources, 4(8), 539-539.