Teknologi Bioflok (Bft) Pada Ikan Lele Betina (Clarias Gariepinus) Selama Periode Rematurasi: Pengaruh Suhu Dan Flok Terhadap Kinerja Reproduksi.
BIOFLOC TECHNOLOGY DURING THE RE-MATURATION
PERIOD OF THE AFRICAN CATFISH (Clarias gariepinus)
FEMALES: EFFECT OF TEMPERATURE AND FLOCS ON
REPRODUCTIVE PERFORMANCE
HASSANE NADIO
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
DECLARATION OF ORIGINALITY
I hereby declare that this thesis titled “BIOFLOC TECHNOLOGY
DURING THE RE-MATURATION PERIOD OF THE AFRICAN CATFISH
(Clarias gariepnus) FEMALES: EFFECT OF TEMPERATURE AND FLOCS
ON REPRODUCTIVE PERFORMANCE” and the work reported herein were
composed by and originated entirely from me under the supervision of my
supervisory committee. I therefore declare that, this is a true copy of my thesis as
approved by my supervisory committee and has not been submitted for a higher
degree to any other University or institution. Information derived from the
published and unpublished work of others has been duly acknowledged in the text
as well as references given in the list of sources.
Bogor, August 2015
Hassane Nadio
Registration No.: C151138721
RINGKASAN
HASSANE NADIO. Teknologi bioflok (BFT) pada ikan lele betina
(Clarias gariepinus) selama periode rematurasi: pengaruh suhu dan flok terhadap
kinerja reproduksi. Dibimbing oleh ODANG CARMAN, WIDANARNI dan
ENANG HARRIS.
Ikan lele merupakan salah satu jenis ikan yang banyak dibudidayakan di
dunia. Namun, proses reproduksinya masih bergantung terhadap musim. Fokus
utama dari setiap pembenihan ikan lele adalah bagaimana menghasilkan jumlah
telur yang banyak dan benih yang berkualitas serta berkelanjutan sepanjang tahun.
Salah satu aspek penting yang berkaitan dengan proses reproduksi adalah
rematurasi. Pada saat musim hujan periode rematurasi pada ikan lele betina
berkisar antara 6 minggu, sedangkan pada musim kemarau berkisar antara 10-14
minggu (FAO, 2008). Oleh karena itu, periode rematurasi ikan lele betina sangat
bervariasi dan menjadi faktor pembatas pada produksi benih skala besar sepanjang
tahun. Biofloc technology (BFT) merupakan salah satu sistem budidaya ikan yang
inovatif. Menurut Avnimelech (2009) pergantian air pada sistem ini dapat
dilakukan secara terbatas atau bahkan tanpa pergantian air. Teknologi ini pertama
kali dikembangkan sebagai alternatif untuk menjawab biosekuritas serta masalah
nutrisi yang mungkin terjadi di kolam. Selain biosekuritas dan aspek nutrisi,
teknologi ini juga terbukti memiliki dampak menguntungkan pada kinerja
reproduksi ikan (termasuk udang).
Tujuan utama dari penelitian ini adalah untuk mengevaluasi efek dari
teknologi bioflok terhadap kinerja reproduksi ikan lele betina, terutama selama
periode rematurasi. Penelitian ini berlangsung selama 7 minggu yang bertempat di
Kolam Percobaan Babakan, Laboratorium Nutrisi Ikan, dan Laboratorium
Lingkungan Akuakultur, Departemen Budidaya Perairan, Fakultas Perikanan dan
Ilmu Kelautan, Institut Pertanian Bogor. Ikan lele betina yang digunakan berbobot
1000±200 gram, dengan jumlah 5 ekor betina per perlakuan. Penelitian ini terdiri
dari 4 perlakuan yaitu perlakuan 1 sebagai kontrol tanpa sistem bioflok
(CW+suhu 25 oC); perlakuan 2 (BFT+suhu 25 oC); perlakuan 3 (BFT+suhu 28
o
C); dan perlakuan 4 (BFT+suhu 31 oC).
Hasil yang terbaik terdapat pada perlakuan 4 dengan indeks kehamilan (IK
≥ 0,48), fekunditas sebanyak 127×103 butir/kg, periode rematurasi pendek
(mencapai 80% ikan betina matang gonad pada minggu ke-3), dan indeks gonadosomatik yang tinggi (GSI ≥ 19%). Sementara hasil yang terendah terdapat pada
perlakuan 1 dengan indeks kehamilan (IK ≤ 0,40), fekunditas sebanyak 57,3×103
butir, periode rematurasi yang lebih lama (mencapai 20% ikan betina matang
gonad pada minggu ke-6) dan GSI yang terendah (GSI ≥ 10%). Dari hasil
penelitian diperoleh hasil berbeda nyata antara perlakuan bioflok dan kontrol.
Namun, tidak ada perbedaan yang signifikan (p>0,05) diantara semua perlakuan
bioflok. Kinerja reproduksi yang lebih baik ditunjukkan oleh ikan betina yang
dipelihara dengan menggunakan sistem BFT dan disarankan teknologi BFT ini
dapat diterapkan pada sistem manajemen induk ikan lele.
Kata kunci : Teknologi Bioflok (BFT), Ikan lele betina, Reproduksi, Re-maturasi
SUMMARY
HASSANE NADIO. Biofloc technology during the re-maturation period of
the African catfish (Clarias gariepinus) Females: effect of temperature and flocs
on reproductive performance. Supervised by ODANG CARMAN,
WIDANARNI and ENANG HARRIS.
The African catfish (Clarias gariepinus) is widely distributed nowadays not
only in Africa but also in many countries in the world. However, its reproduction
strongly depends on seasons. The primary concern of any Clarias gariepinus
hatchery is to understand how to produce a maximum number of high quality eggs
and fry at any time of the year. One of the most important aspects associated with
reproduction is the re-maturation. The re-maturation period of Clarias gariepinus
females range between 6 weeks (FAO, 2008), especially during the rainy season
and up to 10-14 weeks during dry season. Due to such a variation became a
limiting factor for large scale Clarias gariepinus fry production throughout the
year.
Biofloc technology (BFT) is a well-known innovative aquaculture system
based on zero or limited water exchange (Avnimelech, 2009) that is being widely
used in aquaculture nowadays. It was first developed as an alternative to solve not
only biosecurity but also nutritional problems that may occur on the farm. In
addition to its biosecurity and nutritional aspects, the mentioned technology was
also shown to have beneficial impacts on the reproductive performances of some
fish (including shrimp).
The main objective of this study was to evaluate the effects of biofloc
technology on the reproductive performance of Clarias gariepinus females,
especially during their re-maturation period. Thus, females (1000±200g) at a stock
density of 5 females in each treatment were used in a 7 weeks experiment,
involving four treatments: 1 without biofloc at 25˚C (control, CW+25) and the
other three in biofloc systems. The BFT treatments were differentiated by
temperature: biofloc at 25 ˚C (BFT+25), biofloc at 28 ˚C (BFT+28) and biofloc at
31 ˚C (BFT+31). Females reared in the biofloc system at 31˚C achieved better
reproductive performance in terms of pregnancy index (PI ≥ 0.48), higher eggs
production per spawning (127×103) per kg female, shorter re-maturation period
(80% of females reached maturation within 3 weeks), higher gonadal-somatic
index (GSI ≥ 19%) as compared with the control with lower pregnancy index (PI
≤ 0.40), lower eggs production per spawning (57.3×103) per kg female, longer
re-maturation period (20% of females reached maturity within 6 weeks) and
lowest gonadal-somatic index (GSI ≤ 10%) respectively. There was no significant
difference among the BFT treatments (P > 0.05). Thus, the better reproductive
performance showed by females reared in BFT system justified the application of
this technology in Clarias gariepinus broodstock management.
Keywords: Biofloc technology, Catfish females, Reproduction, Re-maturation
© Copyright owned by IPB, 2015
All rights reserved
No part of this document may be reproduced or transmitted in any form or by any
means, electronic, mechanical, photocopying, recording, or otherwise, without
prior written permission from Bogor Agricultural University (IPB)
BIOFLOC TECHNOLOGY DURING THE RE-MATURATION
PERIOD OF THE AFRICAN CATFISH (Clarias gariepinus)
FEMALES: EFFECT OF TEMPERATURE AND FLOCS ON
REPRODUCTIVE PERFORMANCE
HASSANE NADIO
A Thesis
Submitted in partial fulfillment of the requirements
for the degree of
Master of Science
In
Aquaculture
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
External examiner:
Dr Ir Julie Ekasari, MSc
Thesis title
Name
Student Number
Major
: Biofloc Technology during the Re-Maturation Period of
the African catfish (Clarias gariepinus) Females: Effect
of Temperature and Flocs on Reproductive Performance.
: Hassane Nadio
: C151138721
: Aquaculture
Approved by,
Supervisory Committee:
Dr.Ir. Odang Carman, M.Sc
Head-Supervisor
Prof. Dr. Ir. Enang Harris, MS
Co-supervisor
Dr. Ir. Widanarni, M.Si
Co-supervisor
Endorsed by,
Head of Aquaculture Science
Study Program
Dr. Ir. Widanarni, M.Si
Date of examination:
Dean of Graduate School
Dr.Ir. Dahrul Syah, M.Sc. Agr.
Date of Graduation
ACKNOWLEDGEMENT
I am forever grateful to God for the good health and wellbeing that were
necessary to complete this thesis.
I would like to express my special appreciation and thanks to my advisor
Dr.Ir. Odang Carman, you have been a tremendous mentor for me. I would like to
thank you for encouraging my research.
I would also like to thank my committee members, Prof. Dr. Ir. Enang
Harris and Dr. Ir. Widanarni, for serving as my committee members even at
hardship. I also want to thank you for letting my defense be an enjoyable moment,
and for your brilliant comments and suggestions, thanks to you.
I place on record, my sincere thank you to the Government of Indonesia
through the Ministry of Education and Culture for the grant of scholarship.
I take this opportunity to express gratitude to all staff and colleagues in the
Department of Aquaculture, Fisheries and Marine Science Faculty in the Bogor
Agricultural University. I also thank my parents for the unceasing encouragement,
support and attention. I am also grateful to my partner who supported me through
this research.
I also place on record, my sense of gratitude to one and all, who directly or
indirectly, have lent their hand in this research.
Bogor, August 2015
Hassane Nadio
C151138721
TABLE OF CONTENTS
1 INTRODUCTION
Background
1
Problem Statement
2
Objective
3
Hypotheses
3
2 RESEARCH METHODOLOGY
3
Re-Maturation Time
6
Pregnancy Index
6
Gonadal-Somatic Index
6
Fecundity
7
Egg Diameters
7
Fertilization rate
7
Hatching Rate
7
Larvae Survival Rate
7
Biofloc Composition
7
Data analysis
7
3 RESULTS AND DISCUSSION
9
Results
9
Maturity state of females
9
Pregnancy index and Gonadal-somatic index of females
10
Fecundity of females
11
Eggs diameter, Fertilization rate, Hatching rate and larvae survival rateError!
Bookmark not defined.
Feed and flocs quality
13
Water quality parameters
13
Discussion
14
CONCLUSIONS AND RECOMMENDATIONS
16
Conclusion
16
Recommendation
16
REFERENCES
16
LIST OF TABLES
Maturity status of females in BFT+31 at different period of time..................... 9
Maturity status of females in BFT+28 at different period of time..................... 9
Maturity status of females in BFT+25 at different period of time..................... 9
Maturity status of females in CW+25 at different period of time. .................... 9
Mean eggs diameter, hatching rate, fertilization rate and larvae survival
rate of females in different treatments. ....................................................... 12
Proximate composition (% dry weight) of the feed and the flocs. .................. 13
Water quality parameters in each treatment……………………………… .12
LIST OF FIGURES
Body length and abdominal circumference measurements………………6
Females with different Pregnancy index…………………………………6
Mean pregnancy index of catfish females ……………………
………10
Mean gonadal somatic index (GSI, %) ………………………………...11
Mean fecundity (x103)/kg fish of females in each treatment. …...…….12
1 INTRODUCTION
Background
Biofloc technology (BFT) is a well-known innovative aquaculture system
that is being widely used in aquaculture nowadays (Avnimelech 2009). It was first
developed as an alternative system to improve water quality and feed utilization.
Its main principle is to stimulate the growth of microbial flocs that convert
nitrogenous waste from the feed input into microbial biomass (Avnimelech 2009).
This nutritious microbial flocs, composed essentially of free and attached bacteria,
aggregates of particulate organic matter, rotifers and protozoa, are available
continuously in the culture system during the whole farming period as an
additional food source for the cultured organism (Azim and Little 2008; Ballester
et al. 2010; (Emerenciano et al. 2012 and 2013).
In addition to its water quality improvement and nutritional contribution,
BFT was also shown to have beneficial impacts on the reproductive performance
of some cultured species (Emerenciano et al. 2012 and 2013, Ekasari et al. 2013).
Emerenciano et al. (2012 and 2013) suggested that the enhanced reproductive
performance of the blue shrimp (Litopenaeus stylirostris) and the pink shrimp
(Farfantepenaeus duorarum) broodstock in the biofloc systems was attributed to
the utilization of biofloc as an additional feed source for the cultured shrimp.
Furthermore, a similar effect was also observed on the reproductive performance
of the Nile tilapia (Oreochromis niloticus) (Ekasari et al. 2013). The study
showed that the tilapia reared under BFT system showed high cholesterol levels in
the blood, which is known to be a precursor for the biosynthesis of steroid
hormones involved in reproductive processes.
The African catfish (Clarias gariepinus) has been widely distributed not
only in Africa but also in other countries such as Brazil, Indonesia, India, Israel,
Vietnam and the Netherlands. In its natural habitat, it is omnivorous, feeding on
plant materials, plankton, mollusks, fishes, reptiles, and amphibians (Yalcin et al.
2001). The annual reproductive cycle of Clarias gariepinus females consists of a
resting period, a period of gametogenesis and a breeding period. The gonadal
maturation is seasonal and associated with periods of flooding. Thus, it is
influenced by the changes in water temperature, photoperiod and water levels.
However, the increase in water level is the principal factor for triggering their
reproduction (Van der Waal 1974; De Graaf and Janssen 1996). In captivity, the
re-maturation time of the catfish ranges between 6 weeks (FAO 2008), especially
during the rainy season, and up to 12 weeks during dry season. This delay in
reproduction within different seasons can be considered as a limiting factor in
fulfilling the growing market demand through its impact on the seed production.
Therefore, to ensure a continuous supply of seeds, farmers maintain an excess of
broodstock to ensure that egg production goals are met. However, this is
ineffective in terms of higher costs in feed and maintenance of the broodstock.
Two main factors are known to have significant effects on the gonadal
maturation and spawning process in fish i.e. the water temperature and the
nutritional state of females (Barber and Blake 1991; Billard and Breton 1981;
Encina and Granado 1997; Wootton 1990). Water temperature is known to be
2
very important in fish reproduction, since it affects the timing of the maturational
decision and the spawning activity. It also has direct effects on the synthesis and
secretion of hormones influencing gametogenesis (Van Der Kraak and Pankhurst
1997). Niall and Ronald (1995) noted that temperatures above or below the
optimum of 25oC could disturb the reproduction cycle of Clarias gariepinus
females.
Moreover, food availability also has important consequences on the fish
reproduction through its effects on metabolism and surplus of energy (Wootton
1990). Encina and Granado (1997) demonstrated that insufficient quantity and
quality of food supply for the broodstock could either lead to longer time to
maturation or lower egg production. For Clarias gariepinus females, the feeding
level determines the total number of gametes, being highest in better fed females
(Niall and Ronald 1995). Appropriate nutrition not only improves the condition of
the females, but also increases the reproductive success in terms of quality and
quantity of the eggs. Furthermore, Izquierdo et al. (2001) noted that some
specific nutrients such as proteins, lipids, fatty acids, as well as vitamins are
essential nutrients for fish broodstock and their reproduction.
To our knowledge, the effects of biofloc technology application on Clarias
gariepinus females during their re-maturation period on reproductive
performances are still unknown. Thus, this study aims to determine the effect of
maintaining female broodstock in biofloc system on the reproductive performance
of African catfish. In addition, the effects of different temperatures were also
observed.
Problem Statement
The primary concern of any Clarias gariepinus hatchery is to produce the
maximum number of high quality eggs and fry at any time of the year. However,
the seasonality of spawning is a major problem in broodstock management of
African catfish, especially females, which strongly affects their re-maturation
cycle. It considerably imposes both extensive and intensive problem on Clarias
gariepinus farming. One of the solutions that have been used over the past
decades by producers is the maintenance of an excess of broodstock to ensure that
eggs production goals are met at any time of the year. Nevertheless, in intensive
production system, the mentioned solution might increase the production cost of
the fish by increasing the feed cost, since feed is well known to be the most
important part of the production cost. Other methods such as hormonal treatment
are being used for reducing the impact of seasonality on the reproduction cycle of
Clarias gariepinus females, yet, it has to be noticed that in a large scale
production, hormonal treatment may not be the best choice due to its
disadvantages in terms of practice and cost as well. Now and then, the
development of a reliable method in term of controlling the re-maturation cycle of
Clarias gariepinus females that provide a reasonable cost/benefit ratio, to support
economic and sustainability becomes a must for future researches.
3
Objective
The aim of the present study was to evaluate the effects of biofloc
technology at different temperatures on the re-maturation process and
reproductive performance of Clarias gariepinus females.
Hypotheses
1- The biofloc technology could have positive effects on the re-maturation process
of Clarias gariepinus females not only by accelerating the process, but also by
improving the reproductive performance.
2- There could be an optimum temperature that not only promotes the rematuration process of Clarias gariepinus females, but also promotes the
bacterial growth in the biofloc technology.
2 RESEARCH METHODOLOGY
Time and Area of Study
The research was carried out at the teaching farm (Kolam Babakan) of the
Aquaculture Department, Bogor Agricultural University (IPB), Dramaga campus,
West Java province, Indonesia, from January to March 2015.
Experimental Design
Matured females and males Clarias gariepinus between 8 and 9 months
old and weighing 1000g (±200g), reared under common system were used in the 7
weeks experiment, which involved four treatments in a Completely randomized
design: one in a conventional system without biofloc at 25˚C (CW+25) and other
three in a BFT system. The BFT treatments were differentiated by temperature:
biofloc at 25˚C (BFT+25); biofloc at 28˚C (BFT+28) and biofloc at 31˚C
(BFT+31). Each experimental unit comprised of 6 fish (1 male and 6 females),
thus a total of 24 fish (4 males and 20 females) were used and reared in 250 L
conical tank. It had to be emphasized that the replicates used in this experiment
were individual fish, signifying that one female represented one replicates (1
female broodfish = one replicate), thus there were 5 replicates for each treatment.
Feeding were done twice daily using commercial feed (Cargil, 38% protein) at
satiation.
4
Experimental Setup
The experiment was conducted in an air conditioned laboratory with a room
temperature maintained at 25˚C. Conical tanks (250 L) were used as the
experimental units with a constant aeration provided by an air blower. Each tank
was equipped with heaters to maintain the required water temperature. Each
experimental unit comprised of 6 fish (1 male and 5 females), thus a total of 24
fish (4 males and 20 females) were used and reared in 250 L conical tanks. The
replicates used in this experiment were individual fish, thus there were 5 replicates
for each treatment. Males were used for stimulations purposes, since they might
stimulate ovarian growth and development by olfactory and tactile cues (Niall and
Ronald 1995), and therefore; were not the subject of this research.
Biofloc system preparation
To stimulate biofloc growth in the culture water, biofloc systems were
prepared 2 weeks prior to the experiment. Three conical tanks were filled with
250 L water (clear water) and stocked with 20 fish in each tank (±500 g) and
molasses were provided as the carbon source with a C/N ratio of 10 (Avnimelech,
2009). Molasses was added to the biofloc systems on daily basis according to the
formula developed by Avnimelech (2009). After the pre-system was run for two
weeks all fish were removed from the biofloc systems before stocking the tanks
with the experimental fish. Molasses was added to the biofloc systems on daily
basis according to the formula developed by Avnimelech (2009). After the presystem was run for two weeks all fish were removed from the biofloc systems
before stocking the tanks with the experimental fish.
Broodstock Maintenance
Forty mature broodstock kindly provided by the Sukabumi Main Center of
Freshwater Aquaculture Development were brought from Sukabumi and
acclimatized to laboratory conditions for a week. Artificial spawning was
subsequently performed to ensure similar initial reproductive status of the females
prior to the experiment. For this purpose, 20 healthy females were selected and
injected with synthetic analogue of gonadotropin releasing agent and dopamine
inhibitor (Ovaprim, Syndel Lab. Ltd. Canada). Egg stripping was performed 12h
after hormone injection. After stripping, the fish were placed into plastic
containers for recovery (24h).
Tagging was performed (during sampling for initial data) on both the nasal
barbels and the inner mandibular barbels of all the spent females. After the
tagging process, the females were directly put into the experimental system
consisting of 4 treatments.
5
Preparation of spawning facilities
Artificial spawning was performed at the end of the research period (when
females reached maturity) in order to evaluate some reproductive parameters i.e.
fertilization level, hatching level, fecundity, eggs diameter and larval survival.
Artificial spawning was induced by injecting Ovaprim into the dorsal muscle of
20 matured broodstock at a dosage of 0.2 mL per kg of female fish. At the same
time, the males were also injected with 0.1 mL Ovaprim per kg male.
Fertilization and hatching
After observing the 12 hours ovulation period, each female was carefully
removed from the containers and held firmly with a wet towel at both ends. The
abdomen was then pressed carefully to release the eggs into a dry bowl. One gram
of the egg mass of each female, about 750 eggs, was weighed out and mixed with
0.2 mL of milt. The fertilized eggs were then poured into the labeled incubation
plastic containers, previously filled with 1 L of water and incubated at water
temperature range of 25 to 27˚C for hatching.
The fertilized eggs were subsequently incubated in the hatching containers
for a period of 24 to 40 h. The hatching level was estimated 40 h after fertilization,
by counting the number of hatched larvae out of the fertilized eggs.
Larvae survival
After hatching, the unhatched eggs were counted and siphoned out of the
hatching containers. The larvae were daily observed until day 7 to determine the
survival level. No feeding was done during the 7 days.
Water quality
Temperature, dissolved oxygen (DO) and pH were measured every 3 days
using a multi-parameter probe. One liter of water was collected from each
treatment weekly to measure the floc volume (biofloc treatment only), total
ammonical nitrogen (TAN), nitrite (NO2-N) and nitrate (NO3-N). TAN, nitrite-N
and nitrate-N were measured according to the Standard Method for the
Examination of Water and Wastewater (APHA, 1998). Floc volume was
measured based on the sedimentation of the flocs contained in a 50-mL water
sample after 15-20 min (Avnimelech 2009).
6
Observed parameters
Pregnancy Index
The pregnancy index represents the ratio of the fish abdominal
circumference relative to its body length indicating its state of gonadal maturity.
Body length and abdominal circumference were determined using a measuring
tape. The body length that was taken into consideration started from the head of
the fish to its tail (Picture 7A), while the abdominal circumference was measured
the body circumference vertically at the first ray of the dorsal fin (Picture 7B).
A
B
Figure 1 Body length (A) and abdominal circumference measurements (B).
The pregnancy index was then calculated by using the following formula:
�
�
=
�
�
ℎ
A preliminary experiment was conducted to determine the standard
pregnancy index at which the females can be considered as mature and ready for
spawning. We found that pregnancy index of ≥ 0.45 could be considered ideal for
proper spawning and egg production.
Re-Maturation Time
Re-maturation time was calculated by measuring the time it took to a spent
female at the beginning of the research (Figure 5A) to reach the optimum
pregnancy index of 0.45 (Figure 5B) expressed in week.
A
B
C
Figure 2 Females with different Pregnancy index; (A) spent female, (B) 0.45, and
(C) 0.48
Gonadal-Somatic Index
The relationship of gonad weight to the total body weight, or total body
weight to gonad weight, was expressed in percent (Barber and Blake, 1981).
7
� �
=
� ℎ
� ℎ
Fecundity
The fecundity was calculated by weighing 1g of eggs from each female
(triplicate determination were made). The number of eggs in one gram of gonad
was determined by counting in triplicates.
Egg Diameters
This parameter was observed since the egg diameter is of capital
importance for the survival of the larvae. Thus, 30 eggs were collected from the
egg mass of each female after stripping and egg diameter was observed using
microscope.
Fertilization level
The fertilization rate was determined 8h after mixing the eggs and the milt.
For calculating percent of fertilization, a sample of about 750 eggs from each
female from each treatment were carefully placed into a one liter water capacity
container. The fertilization level was calculated using the following formula
(Adebayo, 2006):
��
�
=
No. of fertilized eggs
�
Total No. of eggs counted
ℎ�
=
Number of hatched eggs
�
Total number of eggs
Hatching level
This was carried out by counting the total number of hatched eggs in each
treatment and expressed as a percentage of the total number of eggs.
%ℎ
Larvae Survival level
Percentage survival was determined by counting the total number
of survived larvae one week after hatching.
%
�
=
Number of survived larvae
�
Total number of hatched larvae
Biofloc Composition
The floc composition was determined by analyzing the sediment contained
in a 50-mL water sample collected from each biofloc treatmens and rested for 1520 min in an Imhoff cone (Avnimelech 2012).
Data analysis
One-way analyses of variances (ANOVA) were used to identify significant
differences among the mean values of pregnancy index, GSI, fecundity, eggs
diameter, hatching level and survival of larvae. An ANOVA test was followed by
8
a Duncan’s post-hoc comparison test if significant differences were found. All
statistical analyses were examined at P < 0.05. Correlation coefficient between
the pregnancy index and the GSI was calculated using Pearson’s product-moment
correlation.
9
3 RESULTS AND DISCUSSION
Results
Maturity state of females
The females reared in BFT31 reached maturity 60% faster than the females
in other treatments. After 2 weeks of experimentation, 20% of the females were
matured and within 4 weeks, all females in this treatment had reached maturity
(Table 1). The females reared in BFT28 and BFT25 showed slightly slower
maturity than the broodstock in BFT31, where 20% of the females reached
maturity within 3 weeks. But, all females in biofloc treatments were mature within
4 weeks (Table 2 and 3). On the contrary, the females raised in the conventional
system (CW+25) only started to reach maturity after 6 weeks (Table 4) and all
females in this treatment had achieved maturity after 7 weeks.
Table 1 Maturity status of females in BFT+31 at different period of time.
Treatment
Brooders
1
2
BFT+31
3
4
5
average (% of matured females)
1
NM
NM
NM
NM
NM
0
Maturity status (weeks)
2
3
NM
M
M
M
M
M
M
NM
NM
M
20
80
4
M
M
M
M
M
100
NM= non-matured female, M=matured female ready for spawning, BFT+31= biofloc treatment at
31˚C
Table 2 Maturity status of females in BFT+28 at different period of time.
Treatment
Brooders
1
2
BFT+28
3
4
5
average (% of matured females)
1
NM
NM
NM
NM
NM
0
Maturity status (weeks)
2
3
NM
NM
NM
NM
NM
NM
NM
NM
NM
M
0
20
4
M
M
M
M
M
100
NM= non-matured female, M=matured female ready for spawning, BFT+28= biofloc treatment at
28 ˚C.
Table 3 Maturity status of females in BFT+25 at different period of time.
Treatment
Brooders
1
2
BFT+25
3
4
5
average (% of matured females)
1
NM
NM
NM
NM
NM
0
Maturity status (weeks)
2
3
NM
NM
NM
M
NM
NM
NM
NM
NM
NM
0
20
4
M
M
M
M
M
100
NM= non-matured female, M=matured female ready for spawning, BFT+25= biofloc treatment at
25 ˚C.
Table 4 Maturity status of females in CW+25 at different period of time.
Treatment
Brooders
CW+25
1
2
3
Maturity status (weeks)
1
2
3
NM
NM
NM
NM
NM
NM
NM
NM
NM
4
NM
NM
NM
5
NM
NM
NM
6
NM
NM
NM
7
M
M
M
10
4
5
average (% of matured females)
NM
NM
0
NM
NM
0
NM
NM
0
NM
NM
0
NM
NM
0
M
NM
20
M
M
100
NM= non-matured female, M=matured female ready for spawning, CW= conventional system
without biofloc at 25 ˚C.
Pregnancy index and Gonadal-somatic index of females
There was no significant difference in pregnancy index between treatments
at the beginning of the study (P> 0.05) (Figure 3). However, the pregnancy index
on the 4th week of the experiment showed significant differences between the
biofloc treatments and the conventional system (P < 0.05). The BFT31 had the
highest mean pregnancy index ratio (0.47) compared to other treatments (Figure
3). Among all treatments, CW+25 presented the lowest mean pregnancy index
ratio (0.43). The females placed into the conventional system reached the
optimum pregnancy index ratio within 7 weeks, while the females put into the
biofloc treatments reached the required pregnancy index within only 4 weeks.
Week 0 (initial)
Week 4
0.50
a
Pregnancy index ratio of females
0.48
a
a
0.46
0.44
0.42
0.40
x
x
x
b
x
0.38
0.36
0.34
0.32
0.30
BFT31
BFT28
BFT25
C25
Treatments
Figure 3 Mean pregnancy index of catfish females (n=5) on week 0 and week 4.
BFT31= biofloc treatment at 31˚C, BFT28= biofloc treatment at 28 ˚C,
BFT25= biofloc treatment at 25 ˚C, CW+25= conventional system without
biofloc at 25 ˚C. Different letters above the bars with the same pattern
indicate significant differences (P
PERIOD OF THE AFRICAN CATFISH (Clarias gariepinus)
FEMALES: EFFECT OF TEMPERATURE AND FLOCS ON
REPRODUCTIVE PERFORMANCE
HASSANE NADIO
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
DECLARATION OF ORIGINALITY
I hereby declare that this thesis titled “BIOFLOC TECHNOLOGY
DURING THE RE-MATURATION PERIOD OF THE AFRICAN CATFISH
(Clarias gariepnus) FEMALES: EFFECT OF TEMPERATURE AND FLOCS
ON REPRODUCTIVE PERFORMANCE” and the work reported herein were
composed by and originated entirely from me under the supervision of my
supervisory committee. I therefore declare that, this is a true copy of my thesis as
approved by my supervisory committee and has not been submitted for a higher
degree to any other University or institution. Information derived from the
published and unpublished work of others has been duly acknowledged in the text
as well as references given in the list of sources.
Bogor, August 2015
Hassane Nadio
Registration No.: C151138721
RINGKASAN
HASSANE NADIO. Teknologi bioflok (BFT) pada ikan lele betina
(Clarias gariepinus) selama periode rematurasi: pengaruh suhu dan flok terhadap
kinerja reproduksi. Dibimbing oleh ODANG CARMAN, WIDANARNI dan
ENANG HARRIS.
Ikan lele merupakan salah satu jenis ikan yang banyak dibudidayakan di
dunia. Namun, proses reproduksinya masih bergantung terhadap musim. Fokus
utama dari setiap pembenihan ikan lele adalah bagaimana menghasilkan jumlah
telur yang banyak dan benih yang berkualitas serta berkelanjutan sepanjang tahun.
Salah satu aspek penting yang berkaitan dengan proses reproduksi adalah
rematurasi. Pada saat musim hujan periode rematurasi pada ikan lele betina
berkisar antara 6 minggu, sedangkan pada musim kemarau berkisar antara 10-14
minggu (FAO, 2008). Oleh karena itu, periode rematurasi ikan lele betina sangat
bervariasi dan menjadi faktor pembatas pada produksi benih skala besar sepanjang
tahun. Biofloc technology (BFT) merupakan salah satu sistem budidaya ikan yang
inovatif. Menurut Avnimelech (2009) pergantian air pada sistem ini dapat
dilakukan secara terbatas atau bahkan tanpa pergantian air. Teknologi ini pertama
kali dikembangkan sebagai alternatif untuk menjawab biosekuritas serta masalah
nutrisi yang mungkin terjadi di kolam. Selain biosekuritas dan aspek nutrisi,
teknologi ini juga terbukti memiliki dampak menguntungkan pada kinerja
reproduksi ikan (termasuk udang).
Tujuan utama dari penelitian ini adalah untuk mengevaluasi efek dari
teknologi bioflok terhadap kinerja reproduksi ikan lele betina, terutama selama
periode rematurasi. Penelitian ini berlangsung selama 7 minggu yang bertempat di
Kolam Percobaan Babakan, Laboratorium Nutrisi Ikan, dan Laboratorium
Lingkungan Akuakultur, Departemen Budidaya Perairan, Fakultas Perikanan dan
Ilmu Kelautan, Institut Pertanian Bogor. Ikan lele betina yang digunakan berbobot
1000±200 gram, dengan jumlah 5 ekor betina per perlakuan. Penelitian ini terdiri
dari 4 perlakuan yaitu perlakuan 1 sebagai kontrol tanpa sistem bioflok
(CW+suhu 25 oC); perlakuan 2 (BFT+suhu 25 oC); perlakuan 3 (BFT+suhu 28
o
C); dan perlakuan 4 (BFT+suhu 31 oC).
Hasil yang terbaik terdapat pada perlakuan 4 dengan indeks kehamilan (IK
≥ 0,48), fekunditas sebanyak 127×103 butir/kg, periode rematurasi pendek
(mencapai 80% ikan betina matang gonad pada minggu ke-3), dan indeks gonadosomatik yang tinggi (GSI ≥ 19%). Sementara hasil yang terendah terdapat pada
perlakuan 1 dengan indeks kehamilan (IK ≤ 0,40), fekunditas sebanyak 57,3×103
butir, periode rematurasi yang lebih lama (mencapai 20% ikan betina matang
gonad pada minggu ke-6) dan GSI yang terendah (GSI ≥ 10%). Dari hasil
penelitian diperoleh hasil berbeda nyata antara perlakuan bioflok dan kontrol.
Namun, tidak ada perbedaan yang signifikan (p>0,05) diantara semua perlakuan
bioflok. Kinerja reproduksi yang lebih baik ditunjukkan oleh ikan betina yang
dipelihara dengan menggunakan sistem BFT dan disarankan teknologi BFT ini
dapat diterapkan pada sistem manajemen induk ikan lele.
Kata kunci : Teknologi Bioflok (BFT), Ikan lele betina, Reproduksi, Re-maturasi
SUMMARY
HASSANE NADIO. Biofloc technology during the re-maturation period of
the African catfish (Clarias gariepinus) Females: effect of temperature and flocs
on reproductive performance. Supervised by ODANG CARMAN,
WIDANARNI and ENANG HARRIS.
The African catfish (Clarias gariepinus) is widely distributed nowadays not
only in Africa but also in many countries in the world. However, its reproduction
strongly depends on seasons. The primary concern of any Clarias gariepinus
hatchery is to understand how to produce a maximum number of high quality eggs
and fry at any time of the year. One of the most important aspects associated with
reproduction is the re-maturation. The re-maturation period of Clarias gariepinus
females range between 6 weeks (FAO, 2008), especially during the rainy season
and up to 10-14 weeks during dry season. Due to such a variation became a
limiting factor for large scale Clarias gariepinus fry production throughout the
year.
Biofloc technology (BFT) is a well-known innovative aquaculture system
based on zero or limited water exchange (Avnimelech, 2009) that is being widely
used in aquaculture nowadays. It was first developed as an alternative to solve not
only biosecurity but also nutritional problems that may occur on the farm. In
addition to its biosecurity and nutritional aspects, the mentioned technology was
also shown to have beneficial impacts on the reproductive performances of some
fish (including shrimp).
The main objective of this study was to evaluate the effects of biofloc
technology on the reproductive performance of Clarias gariepinus females,
especially during their re-maturation period. Thus, females (1000±200g) at a stock
density of 5 females in each treatment were used in a 7 weeks experiment,
involving four treatments: 1 without biofloc at 25˚C (control, CW+25) and the
other three in biofloc systems. The BFT treatments were differentiated by
temperature: biofloc at 25 ˚C (BFT+25), biofloc at 28 ˚C (BFT+28) and biofloc at
31 ˚C (BFT+31). Females reared in the biofloc system at 31˚C achieved better
reproductive performance in terms of pregnancy index (PI ≥ 0.48), higher eggs
production per spawning (127×103) per kg female, shorter re-maturation period
(80% of females reached maturation within 3 weeks), higher gonadal-somatic
index (GSI ≥ 19%) as compared with the control with lower pregnancy index (PI
≤ 0.40), lower eggs production per spawning (57.3×103) per kg female, longer
re-maturation period (20% of females reached maturity within 6 weeks) and
lowest gonadal-somatic index (GSI ≤ 10%) respectively. There was no significant
difference among the BFT treatments (P > 0.05). Thus, the better reproductive
performance showed by females reared in BFT system justified the application of
this technology in Clarias gariepinus broodstock management.
Keywords: Biofloc technology, Catfish females, Reproduction, Re-maturation
© Copyright owned by IPB, 2015
All rights reserved
No part of this document may be reproduced or transmitted in any form or by any
means, electronic, mechanical, photocopying, recording, or otherwise, without
prior written permission from Bogor Agricultural University (IPB)
BIOFLOC TECHNOLOGY DURING THE RE-MATURATION
PERIOD OF THE AFRICAN CATFISH (Clarias gariepinus)
FEMALES: EFFECT OF TEMPERATURE AND FLOCS ON
REPRODUCTIVE PERFORMANCE
HASSANE NADIO
A Thesis
Submitted in partial fulfillment of the requirements
for the degree of
Master of Science
In
Aquaculture
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
External examiner:
Dr Ir Julie Ekasari, MSc
Thesis title
Name
Student Number
Major
: Biofloc Technology during the Re-Maturation Period of
the African catfish (Clarias gariepinus) Females: Effect
of Temperature and Flocs on Reproductive Performance.
: Hassane Nadio
: C151138721
: Aquaculture
Approved by,
Supervisory Committee:
Dr.Ir. Odang Carman, M.Sc
Head-Supervisor
Prof. Dr. Ir. Enang Harris, MS
Co-supervisor
Dr. Ir. Widanarni, M.Si
Co-supervisor
Endorsed by,
Head of Aquaculture Science
Study Program
Dr. Ir. Widanarni, M.Si
Date of examination:
Dean of Graduate School
Dr.Ir. Dahrul Syah, M.Sc. Agr.
Date of Graduation
ACKNOWLEDGEMENT
I am forever grateful to God for the good health and wellbeing that were
necessary to complete this thesis.
I would like to express my special appreciation and thanks to my advisor
Dr.Ir. Odang Carman, you have been a tremendous mentor for me. I would like to
thank you for encouraging my research.
I would also like to thank my committee members, Prof. Dr. Ir. Enang
Harris and Dr. Ir. Widanarni, for serving as my committee members even at
hardship. I also want to thank you for letting my defense be an enjoyable moment,
and for your brilliant comments and suggestions, thanks to you.
I place on record, my sincere thank you to the Government of Indonesia
through the Ministry of Education and Culture for the grant of scholarship.
I take this opportunity to express gratitude to all staff and colleagues in the
Department of Aquaculture, Fisheries and Marine Science Faculty in the Bogor
Agricultural University. I also thank my parents for the unceasing encouragement,
support and attention. I am also grateful to my partner who supported me through
this research.
I also place on record, my sense of gratitude to one and all, who directly or
indirectly, have lent their hand in this research.
Bogor, August 2015
Hassane Nadio
C151138721
TABLE OF CONTENTS
1 INTRODUCTION
Background
1
Problem Statement
2
Objective
3
Hypotheses
3
2 RESEARCH METHODOLOGY
3
Re-Maturation Time
6
Pregnancy Index
6
Gonadal-Somatic Index
6
Fecundity
7
Egg Diameters
7
Fertilization rate
7
Hatching Rate
7
Larvae Survival Rate
7
Biofloc Composition
7
Data analysis
7
3 RESULTS AND DISCUSSION
9
Results
9
Maturity state of females
9
Pregnancy index and Gonadal-somatic index of females
10
Fecundity of females
11
Eggs diameter, Fertilization rate, Hatching rate and larvae survival rateError!
Bookmark not defined.
Feed and flocs quality
13
Water quality parameters
13
Discussion
14
CONCLUSIONS AND RECOMMENDATIONS
16
Conclusion
16
Recommendation
16
REFERENCES
16
LIST OF TABLES
Maturity status of females in BFT+31 at different period of time..................... 9
Maturity status of females in BFT+28 at different period of time..................... 9
Maturity status of females in BFT+25 at different period of time..................... 9
Maturity status of females in CW+25 at different period of time. .................... 9
Mean eggs diameter, hatching rate, fertilization rate and larvae survival
rate of females in different treatments. ....................................................... 12
Proximate composition (% dry weight) of the feed and the flocs. .................. 13
Water quality parameters in each treatment……………………………… .12
LIST OF FIGURES
Body length and abdominal circumference measurements………………6
Females with different Pregnancy index…………………………………6
Mean pregnancy index of catfish females ……………………
………10
Mean gonadal somatic index (GSI, %) ………………………………...11
Mean fecundity (x103)/kg fish of females in each treatment. …...…….12
1 INTRODUCTION
Background
Biofloc technology (BFT) is a well-known innovative aquaculture system
that is being widely used in aquaculture nowadays (Avnimelech 2009). It was first
developed as an alternative system to improve water quality and feed utilization.
Its main principle is to stimulate the growth of microbial flocs that convert
nitrogenous waste from the feed input into microbial biomass (Avnimelech 2009).
This nutritious microbial flocs, composed essentially of free and attached bacteria,
aggregates of particulate organic matter, rotifers and protozoa, are available
continuously in the culture system during the whole farming period as an
additional food source for the cultured organism (Azim and Little 2008; Ballester
et al. 2010; (Emerenciano et al. 2012 and 2013).
In addition to its water quality improvement and nutritional contribution,
BFT was also shown to have beneficial impacts on the reproductive performance
of some cultured species (Emerenciano et al. 2012 and 2013, Ekasari et al. 2013).
Emerenciano et al. (2012 and 2013) suggested that the enhanced reproductive
performance of the blue shrimp (Litopenaeus stylirostris) and the pink shrimp
(Farfantepenaeus duorarum) broodstock in the biofloc systems was attributed to
the utilization of biofloc as an additional feed source for the cultured shrimp.
Furthermore, a similar effect was also observed on the reproductive performance
of the Nile tilapia (Oreochromis niloticus) (Ekasari et al. 2013). The study
showed that the tilapia reared under BFT system showed high cholesterol levels in
the blood, which is known to be a precursor for the biosynthesis of steroid
hormones involved in reproductive processes.
The African catfish (Clarias gariepinus) has been widely distributed not
only in Africa but also in other countries such as Brazil, Indonesia, India, Israel,
Vietnam and the Netherlands. In its natural habitat, it is omnivorous, feeding on
plant materials, plankton, mollusks, fishes, reptiles, and amphibians (Yalcin et al.
2001). The annual reproductive cycle of Clarias gariepinus females consists of a
resting period, a period of gametogenesis and a breeding period. The gonadal
maturation is seasonal and associated with periods of flooding. Thus, it is
influenced by the changes in water temperature, photoperiod and water levels.
However, the increase in water level is the principal factor for triggering their
reproduction (Van der Waal 1974; De Graaf and Janssen 1996). In captivity, the
re-maturation time of the catfish ranges between 6 weeks (FAO 2008), especially
during the rainy season, and up to 12 weeks during dry season. This delay in
reproduction within different seasons can be considered as a limiting factor in
fulfilling the growing market demand through its impact on the seed production.
Therefore, to ensure a continuous supply of seeds, farmers maintain an excess of
broodstock to ensure that egg production goals are met. However, this is
ineffective in terms of higher costs in feed and maintenance of the broodstock.
Two main factors are known to have significant effects on the gonadal
maturation and spawning process in fish i.e. the water temperature and the
nutritional state of females (Barber and Blake 1991; Billard and Breton 1981;
Encina and Granado 1997; Wootton 1990). Water temperature is known to be
2
very important in fish reproduction, since it affects the timing of the maturational
decision and the spawning activity. It also has direct effects on the synthesis and
secretion of hormones influencing gametogenesis (Van Der Kraak and Pankhurst
1997). Niall and Ronald (1995) noted that temperatures above or below the
optimum of 25oC could disturb the reproduction cycle of Clarias gariepinus
females.
Moreover, food availability also has important consequences on the fish
reproduction through its effects on metabolism and surplus of energy (Wootton
1990). Encina and Granado (1997) demonstrated that insufficient quantity and
quality of food supply for the broodstock could either lead to longer time to
maturation or lower egg production. For Clarias gariepinus females, the feeding
level determines the total number of gametes, being highest in better fed females
(Niall and Ronald 1995). Appropriate nutrition not only improves the condition of
the females, but also increases the reproductive success in terms of quality and
quantity of the eggs. Furthermore, Izquierdo et al. (2001) noted that some
specific nutrients such as proteins, lipids, fatty acids, as well as vitamins are
essential nutrients for fish broodstock and their reproduction.
To our knowledge, the effects of biofloc technology application on Clarias
gariepinus females during their re-maturation period on reproductive
performances are still unknown. Thus, this study aims to determine the effect of
maintaining female broodstock in biofloc system on the reproductive performance
of African catfish. In addition, the effects of different temperatures were also
observed.
Problem Statement
The primary concern of any Clarias gariepinus hatchery is to produce the
maximum number of high quality eggs and fry at any time of the year. However,
the seasonality of spawning is a major problem in broodstock management of
African catfish, especially females, which strongly affects their re-maturation
cycle. It considerably imposes both extensive and intensive problem on Clarias
gariepinus farming. One of the solutions that have been used over the past
decades by producers is the maintenance of an excess of broodstock to ensure that
eggs production goals are met at any time of the year. Nevertheless, in intensive
production system, the mentioned solution might increase the production cost of
the fish by increasing the feed cost, since feed is well known to be the most
important part of the production cost. Other methods such as hormonal treatment
are being used for reducing the impact of seasonality on the reproduction cycle of
Clarias gariepinus females, yet, it has to be noticed that in a large scale
production, hormonal treatment may not be the best choice due to its
disadvantages in terms of practice and cost as well. Now and then, the
development of a reliable method in term of controlling the re-maturation cycle of
Clarias gariepinus females that provide a reasonable cost/benefit ratio, to support
economic and sustainability becomes a must for future researches.
3
Objective
The aim of the present study was to evaluate the effects of biofloc
technology at different temperatures on the re-maturation process and
reproductive performance of Clarias gariepinus females.
Hypotheses
1- The biofloc technology could have positive effects on the re-maturation process
of Clarias gariepinus females not only by accelerating the process, but also by
improving the reproductive performance.
2- There could be an optimum temperature that not only promotes the rematuration process of Clarias gariepinus females, but also promotes the
bacterial growth in the biofloc technology.
2 RESEARCH METHODOLOGY
Time and Area of Study
The research was carried out at the teaching farm (Kolam Babakan) of the
Aquaculture Department, Bogor Agricultural University (IPB), Dramaga campus,
West Java province, Indonesia, from January to March 2015.
Experimental Design
Matured females and males Clarias gariepinus between 8 and 9 months
old and weighing 1000g (±200g), reared under common system were used in the 7
weeks experiment, which involved four treatments in a Completely randomized
design: one in a conventional system without biofloc at 25˚C (CW+25) and other
three in a BFT system. The BFT treatments were differentiated by temperature:
biofloc at 25˚C (BFT+25); biofloc at 28˚C (BFT+28) and biofloc at 31˚C
(BFT+31). Each experimental unit comprised of 6 fish (1 male and 6 females),
thus a total of 24 fish (4 males and 20 females) were used and reared in 250 L
conical tank. It had to be emphasized that the replicates used in this experiment
were individual fish, signifying that one female represented one replicates (1
female broodfish = one replicate), thus there were 5 replicates for each treatment.
Feeding were done twice daily using commercial feed (Cargil, 38% protein) at
satiation.
4
Experimental Setup
The experiment was conducted in an air conditioned laboratory with a room
temperature maintained at 25˚C. Conical tanks (250 L) were used as the
experimental units with a constant aeration provided by an air blower. Each tank
was equipped with heaters to maintain the required water temperature. Each
experimental unit comprised of 6 fish (1 male and 5 females), thus a total of 24
fish (4 males and 20 females) were used and reared in 250 L conical tanks. The
replicates used in this experiment were individual fish, thus there were 5 replicates
for each treatment. Males were used for stimulations purposes, since they might
stimulate ovarian growth and development by olfactory and tactile cues (Niall and
Ronald 1995), and therefore; were not the subject of this research.
Biofloc system preparation
To stimulate biofloc growth in the culture water, biofloc systems were
prepared 2 weeks prior to the experiment. Three conical tanks were filled with
250 L water (clear water) and stocked with 20 fish in each tank (±500 g) and
molasses were provided as the carbon source with a C/N ratio of 10 (Avnimelech,
2009). Molasses was added to the biofloc systems on daily basis according to the
formula developed by Avnimelech (2009). After the pre-system was run for two
weeks all fish were removed from the biofloc systems before stocking the tanks
with the experimental fish. Molasses was added to the biofloc systems on daily
basis according to the formula developed by Avnimelech (2009). After the presystem was run for two weeks all fish were removed from the biofloc systems
before stocking the tanks with the experimental fish.
Broodstock Maintenance
Forty mature broodstock kindly provided by the Sukabumi Main Center of
Freshwater Aquaculture Development were brought from Sukabumi and
acclimatized to laboratory conditions for a week. Artificial spawning was
subsequently performed to ensure similar initial reproductive status of the females
prior to the experiment. For this purpose, 20 healthy females were selected and
injected with synthetic analogue of gonadotropin releasing agent and dopamine
inhibitor (Ovaprim, Syndel Lab. Ltd. Canada). Egg stripping was performed 12h
after hormone injection. After stripping, the fish were placed into plastic
containers for recovery (24h).
Tagging was performed (during sampling for initial data) on both the nasal
barbels and the inner mandibular barbels of all the spent females. After the
tagging process, the females were directly put into the experimental system
consisting of 4 treatments.
5
Preparation of spawning facilities
Artificial spawning was performed at the end of the research period (when
females reached maturity) in order to evaluate some reproductive parameters i.e.
fertilization level, hatching level, fecundity, eggs diameter and larval survival.
Artificial spawning was induced by injecting Ovaprim into the dorsal muscle of
20 matured broodstock at a dosage of 0.2 mL per kg of female fish. At the same
time, the males were also injected with 0.1 mL Ovaprim per kg male.
Fertilization and hatching
After observing the 12 hours ovulation period, each female was carefully
removed from the containers and held firmly with a wet towel at both ends. The
abdomen was then pressed carefully to release the eggs into a dry bowl. One gram
of the egg mass of each female, about 750 eggs, was weighed out and mixed with
0.2 mL of milt. The fertilized eggs were then poured into the labeled incubation
plastic containers, previously filled with 1 L of water and incubated at water
temperature range of 25 to 27˚C for hatching.
The fertilized eggs were subsequently incubated in the hatching containers
for a period of 24 to 40 h. The hatching level was estimated 40 h after fertilization,
by counting the number of hatched larvae out of the fertilized eggs.
Larvae survival
After hatching, the unhatched eggs were counted and siphoned out of the
hatching containers. The larvae were daily observed until day 7 to determine the
survival level. No feeding was done during the 7 days.
Water quality
Temperature, dissolved oxygen (DO) and pH were measured every 3 days
using a multi-parameter probe. One liter of water was collected from each
treatment weekly to measure the floc volume (biofloc treatment only), total
ammonical nitrogen (TAN), nitrite (NO2-N) and nitrate (NO3-N). TAN, nitrite-N
and nitrate-N were measured according to the Standard Method for the
Examination of Water and Wastewater (APHA, 1998). Floc volume was
measured based on the sedimentation of the flocs contained in a 50-mL water
sample after 15-20 min (Avnimelech 2009).
6
Observed parameters
Pregnancy Index
The pregnancy index represents the ratio of the fish abdominal
circumference relative to its body length indicating its state of gonadal maturity.
Body length and abdominal circumference were determined using a measuring
tape. The body length that was taken into consideration started from the head of
the fish to its tail (Picture 7A), while the abdominal circumference was measured
the body circumference vertically at the first ray of the dorsal fin (Picture 7B).
A
B
Figure 1 Body length (A) and abdominal circumference measurements (B).
The pregnancy index was then calculated by using the following formula:
�
�
=
�
�
ℎ
A preliminary experiment was conducted to determine the standard
pregnancy index at which the females can be considered as mature and ready for
spawning. We found that pregnancy index of ≥ 0.45 could be considered ideal for
proper spawning and egg production.
Re-Maturation Time
Re-maturation time was calculated by measuring the time it took to a spent
female at the beginning of the research (Figure 5A) to reach the optimum
pregnancy index of 0.45 (Figure 5B) expressed in week.
A
B
C
Figure 2 Females with different Pregnancy index; (A) spent female, (B) 0.45, and
(C) 0.48
Gonadal-Somatic Index
The relationship of gonad weight to the total body weight, or total body
weight to gonad weight, was expressed in percent (Barber and Blake, 1981).
7
� �
=
� ℎ
� ℎ
Fecundity
The fecundity was calculated by weighing 1g of eggs from each female
(triplicate determination were made). The number of eggs in one gram of gonad
was determined by counting in triplicates.
Egg Diameters
This parameter was observed since the egg diameter is of capital
importance for the survival of the larvae. Thus, 30 eggs were collected from the
egg mass of each female after stripping and egg diameter was observed using
microscope.
Fertilization level
The fertilization rate was determined 8h after mixing the eggs and the milt.
For calculating percent of fertilization, a sample of about 750 eggs from each
female from each treatment were carefully placed into a one liter water capacity
container. The fertilization level was calculated using the following formula
(Adebayo, 2006):
��
�
=
No. of fertilized eggs
�
Total No. of eggs counted
ℎ�
=
Number of hatched eggs
�
Total number of eggs
Hatching level
This was carried out by counting the total number of hatched eggs in each
treatment and expressed as a percentage of the total number of eggs.
%ℎ
Larvae Survival level
Percentage survival was determined by counting the total number
of survived larvae one week after hatching.
%
�
=
Number of survived larvae
�
Total number of hatched larvae
Biofloc Composition
The floc composition was determined by analyzing the sediment contained
in a 50-mL water sample collected from each biofloc treatmens and rested for 1520 min in an Imhoff cone (Avnimelech 2012).
Data analysis
One-way analyses of variances (ANOVA) were used to identify significant
differences among the mean values of pregnancy index, GSI, fecundity, eggs
diameter, hatching level and survival of larvae. An ANOVA test was followed by
8
a Duncan’s post-hoc comparison test if significant differences were found. All
statistical analyses were examined at P < 0.05. Correlation coefficient between
the pregnancy index and the GSI was calculated using Pearson’s product-moment
correlation.
9
3 RESULTS AND DISCUSSION
Results
Maturity state of females
The females reared in BFT31 reached maturity 60% faster than the females
in other treatments. After 2 weeks of experimentation, 20% of the females were
matured and within 4 weeks, all females in this treatment had reached maturity
(Table 1). The females reared in BFT28 and BFT25 showed slightly slower
maturity than the broodstock in BFT31, where 20% of the females reached
maturity within 3 weeks. But, all females in biofloc treatments were mature within
4 weeks (Table 2 and 3). On the contrary, the females raised in the conventional
system (CW+25) only started to reach maturity after 6 weeks (Table 4) and all
females in this treatment had achieved maturity after 7 weeks.
Table 1 Maturity status of females in BFT+31 at different period of time.
Treatment
Brooders
1
2
BFT+31
3
4
5
average (% of matured females)
1
NM
NM
NM
NM
NM
0
Maturity status (weeks)
2
3
NM
M
M
M
M
M
M
NM
NM
M
20
80
4
M
M
M
M
M
100
NM= non-matured female, M=matured female ready for spawning, BFT+31= biofloc treatment at
31˚C
Table 2 Maturity status of females in BFT+28 at different period of time.
Treatment
Brooders
1
2
BFT+28
3
4
5
average (% of matured females)
1
NM
NM
NM
NM
NM
0
Maturity status (weeks)
2
3
NM
NM
NM
NM
NM
NM
NM
NM
NM
M
0
20
4
M
M
M
M
M
100
NM= non-matured female, M=matured female ready for spawning, BFT+28= biofloc treatment at
28 ˚C.
Table 3 Maturity status of females in BFT+25 at different period of time.
Treatment
Brooders
1
2
BFT+25
3
4
5
average (% of matured females)
1
NM
NM
NM
NM
NM
0
Maturity status (weeks)
2
3
NM
NM
NM
M
NM
NM
NM
NM
NM
NM
0
20
4
M
M
M
M
M
100
NM= non-matured female, M=matured female ready for spawning, BFT+25= biofloc treatment at
25 ˚C.
Table 4 Maturity status of females in CW+25 at different period of time.
Treatment
Brooders
CW+25
1
2
3
Maturity status (weeks)
1
2
3
NM
NM
NM
NM
NM
NM
NM
NM
NM
4
NM
NM
NM
5
NM
NM
NM
6
NM
NM
NM
7
M
M
M
10
4
5
average (% of matured females)
NM
NM
0
NM
NM
0
NM
NM
0
NM
NM
0
NM
NM
0
M
NM
20
M
M
100
NM= non-matured female, M=matured female ready for spawning, CW= conventional system
without biofloc at 25 ˚C.
Pregnancy index and Gonadal-somatic index of females
There was no significant difference in pregnancy index between treatments
at the beginning of the study (P> 0.05) (Figure 3). However, the pregnancy index
on the 4th week of the experiment showed significant differences between the
biofloc treatments and the conventional system (P < 0.05). The BFT31 had the
highest mean pregnancy index ratio (0.47) compared to other treatments (Figure
3). Among all treatments, CW+25 presented the lowest mean pregnancy index
ratio (0.43). The females placed into the conventional system reached the
optimum pregnancy index ratio within 7 weeks, while the females put into the
biofloc treatments reached the required pregnancy index within only 4 weeks.
Week 0 (initial)
Week 4
0.50
a
Pregnancy index ratio of females
0.48
a
a
0.46
0.44
0.42
0.40
x
x
x
b
x
0.38
0.36
0.34
0.32
0.30
BFT31
BFT28
BFT25
C25
Treatments
Figure 3 Mean pregnancy index of catfish females (n=5) on week 0 and week 4.
BFT31= biofloc treatment at 31˚C, BFT28= biofloc treatment at 28 ˚C,
BFT25= biofloc treatment at 25 ˚C, CW+25= conventional system without
biofloc at 25 ˚C. Different letters above the bars with the same pattern
indicate significant differences (P