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This study deals with investigations on how to control off-season maturation and spawning of the Pacific white shrimp Litopenaeus vannamei by using various maturation techniques. For the experiment, the broodstock were separated into five groups (Group 1: Control, Group 2: Serotonin-injected, Group 3: Ablated, Group 4: Temperature-fluctuated, and Group 5: Another ablated groups). Each of the first four groups were stocked into a 2-m diameter round tank at density of 9.44 shrimps per m 2 (2:1, female/male), while Group 5 were stocked into a 3-m diameter tank at density of 5.67 shrimps per m 2 (1:1, female/male). The experiment continued for 2 months until maturation in a recirculation system. Each female was tagged and any ripe female carrying a spermatophore was removed to spawn individually in a spawning tank. The first spawnings occurred on 25-28 th days of the experiment in all the groups. The highest female spawning rate (55-90%) and fecundity (79,778-125,015 eggs) were obtained in the eyestalk-ablated groups (P<0.05). Serotonin (Group 2) induced ovarium development in 35% of the females, generating 60,277 eggs per female. Cyclic temperature fluctuation (Group 4) stimulated ovarium maturation in 39% of the females with a mean fecundity of 28,500 eggs per female (P<0.05). Mean egg fertility rates ranged from 63.08% to 96%, and hatching rates from 8.53% to 31%. Spawning, fecundity and hatching rates were found to be different between the two eyestalk-ablated groups (Group 3 and 5), and the reasons were thought to be due to tank size and/or shrimp stocking density. Our broodstock displayed poor reproductive performance with abnormal egg morphology and low egg hatching rates. The stress caused by off-season reproduction and low genetic variation due to past selective breeding programs might have seriously hampered the reproductive performance of our broodstock. The results of this study has demonstrated that, under M editerranean climatic conditions, the broodstock of this non-indigenous shrimp species can be readily matured and spawned out of season in recirculating systems.
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Turkish Journal of Fisheries and Aquatic Sciences 11: 15-23 (2011)
www.trjfas.org
ISSN 1303-2712
DOI: 10.4194/trjfas.2011.0103
© Published by Central Fisheries Research Institute (CFRI) Trabzon, Turkey
in cooperation with Japan International Cooperation Agency (JICA), Japan
Off-season Maturation and Spawning of the Pacific White Shrimp
Litopenaeus vannamei in Sub-tropical Conditions
Metin Kumlu1,*, Serhat Türkmen1, Mehmet Kumlu1, O. Tufan Eroldoğan1
1 Çukurova University, Faculty of Fisheries, 01330 Balcalı, Adana, T urkey .
Received 26 March 2010
Accept ed 23 September 2010
Abstract
This study deals with investigations on how to control off-season maturation and spawning of the Pacific white shrimp
Litopenaeus vannamei by using various maturation techniques. For the experiment, the broodstock were separated into five groups
(Group 1: Control, Group 2: Serotonin-injected, Group 3: Ablated, Group 4: Temperature-fluctuated, and Group 5: Another ablated
groups). Each of the first four groups were stocked into a 2-m diameter round tank at density of 9.44 shrimps per m2 (2:1, female/male),
while Group 5 were stocked into a 3-m diameter tank at density of 5.67 shrimps per m2 (1:1, female/male). The experiment continued for
2 months until maturation in a recirculation system. Each female was tagged and any ripe female carrying a spermatophore was removed
to spawn individually in a spawning tank. The first spawnings occurred on 25-28th days of the experiment in all the groups. The highest
female spawning rate (55-90%) and fecundity (79,778-125,015 eggs) were obtained in the eyestalk-ablated groups (P<0.05). Serotonin
(Group 2) induced ovarium development in 35% of the females, generating 60,277 eggs per female. Cyclic temperature fluctuation
(Group 4) stimulated ovarium maturation in 39% of the females with a mean fecundity of 28,500 eggs per female (P<0.05). Mean egg
fertility rates ranged from 63.08% to 96%, and hatching rates from 8.53% to 31%. Spawning, fecundity and hatching rates were found to
be different between the two eyestalk-ablated groups (Group 3 and 5), and the reasons were thought to be due to tank size and/or shrimp
stocking density. Our broodstock displayed poor reproductive performance with abnormal egg morphology and low egg hatching rates.
The stress caused by off-season reproduction and low genetic variation due to past selective breeding programs might have seriously
hampered the reproductive performance of our broodstock. The results of this study has demonstrated that, under Mediterranean climatic
conditions, the broodstock of this non-indigenous shrimp species can be readily matured and spawned out of season in recirculating
systems.
Keywords: Shrimp, Litopenaeus vannamei, reproduction, maturation, spawning, egg.
Pasifik Beyaz Karidesi (Litopenaeus vannamei)’nin Yarı-tropik İklim Koşullarında Mevsim-dışı
Olgunlaştırılıp Yumurtlatılması
Özet
Bu çalışma, Pasifik bey az karidesi Litopenaeus vannamei’nin fark teknikler kullanılarak mevsim-şı olgunlaştırılması ve
yumurtlatılması ile ilgili araştırmaları kapsamaktadır. Deneme için anaçlar, 5 ayrı gruba (1. Grup: Kontrol, 2. Grup: Serotonin enjekte
edilenler, 3. Grup:zsapı kesilenler, 4. Grup: Sıcak/soğuk dalgalanması uygulananlar ve 5. Grup: Diğer gözsapı kesilenler) ayrılmıştır.
İlk dört grubun her biri 2-m çapında birer adet yuvarlak tanka m2ye 9,44 karides (2:1, dişi/erkek oranı), 5. Grup ise 3-m çap ında bir tanka
m2ye 5,67 karides (1:1, dişi/erkek ora) olacak şekilde stoklanmışlardır. Anaçlar; kulkahanede kurulan bir resirküle sistemde 2 ay
süren deneme boyunca olgunlaştırılmışlardır. Her dişi karides markalanmış ve olgun-spermatofor taşıy an dişiler anaç tanklarından
yumurtlama tanklarına alınarak bireysel olarak yumurtlatılmışlardır. Tüm gruplarda da ilk yumurtlamalar denemenin 25-28. günlerinde
elde edilmiştir. En yüksek dişi yumurtlama oranı (%55-90) ve yumurta verimlili (79.778-125.015 adet) zsapı kesilen gruplarda
kaydedilmiştir (P<0,05). Serotonin uygulaması (Grup 2) dişilerin %35’inde ovaryum gelişimini uyarmış ve bu grupta her dişi ortalama
60.277 yumurta üretmiştir. Deneme boy unca caklık dalgalanması (Grup 4) dişilerin %39’unda ovaryum gelişimini uyarmış ve bu
grupta ortalama dişi başına 28.500 adet yumurta elde edilmiştir (P<0,05). Tüm gruplarda yumurta döllülük oranı %63,08 ile %96, açılma
oranı ise %8,53 ile %31 arasında çıkmıştır. Gözsap ı kesimi y apılan gruplarda (Grup 3 ve 5) yumurtlama oranı, yumurta verimlili ve
açılma oranı açından önemli farklılıklar belirlenmiş ve bunun nedenlerinin tank yüklüğü, anaç stok ora ve cinsiyet orandan
kaynaklandığı yargısına varılmıştır. Anaçlarımız, anormal yumurta morfolojisi veşük açılma oranları şeklinde kendini gösteren zayıf
bir üreme performansı sergilemişlerdir. Anaçların mevsim-şı üremeye zorlanmaları neticesinde ortaya çıkan stresin ve ayrıca
anaçlardaki şük genetik varyasyonun yumurta kalitesini düşürerek yumurta açılma oranı olumsuz etkilediği sonucuna varılmıştır. Bu
çalışma L. vannameinin Akdeniz iklim koşullarında resirküle sistemler kullanılarak özelliklezsapı kesim tekniğiyle mevsim dışında
rahatlıkla olgunlaştırılıp yumurtlatılabileceğini göstermtir.
Anahtar kelimeler: Karides, Litopenaeus vannamei, üreme, olgunlaştırma, yumurtlatma, yumurta.
Anahtar Kelimeler:
16
M. Kumlu et al. / Turk. J. Fish. Aquat. Sci. 11: 15-23 (2011)
Introduction
Availability of gravid broodstock in the wild is
possible throughout the year in tropical countries, but
mature females can only be obtained during certain
seasons in the sub-tropical Mediterranean countries (Aktaş
and Kumlu, 2003). In such countries, the broodstock have
to be matured and spawned out of season in temperature-
controlled recirculation systems in captivity (Kumlu et al.,
2003). Off-season maturation and spawning, that would
allow larval and nursery culture of shrimps to be carried
out in greenhouses in winter months, can provide
opportunity to extend production season of shrimp
farming to a few months in temperate regions.
In the last decade, farming of the Pacific white
shrimp Litopenaeus vannamei, of which fast growing and
disease resistant strains have been developed by selective
breeding programs, has been expanding throughout the
world, especially in the far-eastern countries such as
Thailand, Vietnam, Indonesia, China and India. This
species can be readily reproduced in captivity, has wide
tolerance to environmental parameters, better utilizes
low-protein containing diets, and grows fast compared to
other penaeid shrimp species (Wyban, 2007). It is well
known that shrimp growth is fast during the warm season
(25-34C) between May and October (5-6 months) in the
Mediterranean region (Kumlu et al., 2003; Kumlu and
Kır, 2005). If this species is to be cultured sucessfuly in
the subtropical regions of Turkey, its reproduction has to
be fully controlled and its seed production must be shifted
towards winter or early-spring in the year. We carried out
several investigations on off-season maturation and
spawning (Aktaş and Kumlu, 1999; Aktaş et al., 2003),
and over-wintering possibilities of the green tiger shrimp
(Penaeus semisulcatus), a native species to North-eastern
Mediterranean (Kır and Kumlu, 2006; 2008a,b).
However, no research has been yet conducted in detail to
obtain knowledge on reproduction performance of off-
season maturation and spawning of L. vannamei in sub-
tropical conditions.
Induction of maturation through eyestalk ablation
technique is extensivelly used by almost all commercial
hatcheries and research facilities worldwide (Caillouet,
1973; Muthu and Laximinarayana, 1977; Lumare, 1979;
Hillier, 1984; Browdy and Samocha, 1985; Browdy,
1992; Aktaş and Kumlu, 1999). Despite its several
advantages, this technique produce poorer quality larvae
after successive spawnings and the spawners have to be
discarded due to rapid loss of condition (Emmerson, 1980;
Chamberlain, 1985; Primavera, 1985; Makinouchi ve
Primavera, 1987; Aktaş and Kumlu, 1999). Alternative
techniques to control shrimp reproduction have received
little attention, and such studies have mainly concentrated
on the injection of various hormones or manipulations of
temperature/photoperiod regimes.
Serotonin (5-hydroxytryptamine; 5-HT), a
neurotransmitter, has shown to induce reproduction in the
crayfish Procambarus clarkii and the lobster Homarus
americanus (Kulkarni et al., 1992; Fingerman, 1997), the
giant freshwater shrimp Macrobrachium rosenbergii
(Meeratana et al., 2006), and some penaeid shrimps such
as P. monodon (Wongprasert et al., 2006), L. vannamei,
L. stylirostris (Vaca and Alfaro, 2000; Alfaro et al., 2004)
as well as P. semisulcatus (Aktaş and Kumlu, 2005).
Serotonin is reported to inhibit GIH (gonad inhibiting
hormone), secreted from the X-organ/sinus complex, or
stimulate GSH (gonad stimulating hormone) in decapod
crustaceans (Sarojini et al., 1995; Fingerman, 1997;
Tinikul et al., 2008). Recently, it has been reported that
GnRH plays an important role in the development of
ovarium in P. monodon Ngernsoungnern et al., 2008), and
serotonin induces ovarian maturation by increasing
vitellogenin accumulation in Fenneropenaeus indicus
(Santoshi et al., 2009). Similarly, progesteron was also
found to influence ovarian development and spawning in
Metapenaeus ensis (Yano, 1985), and improve sperm
quality in L. vannamei (Alfaro, 1996). Yano and Wyban
(1993) stated that HCG stimulates reproduction in
shrimps, while Aktaş and Kumlu (2005) found unclear
effects of HCG and LHRH-a on the reproduction of P.
semisulcatus. Up to now, no study has shown the effects
of such hormones on off-season maturation and spawning
in L. vannamei.
It is well known that some environmental factors
have effects on reproductive performance of penaeid
shrimps in hatcheries. In general, long photoperiods and
high temperatures were reported to be required for
reproduction in P. japonicus (Laubier-Bonichon, 1978), P.
setiferus (Brown et al., 1979), P. esculentus (Crocos and
Kerr, 1986) and P. duorarum (Cripe, 1994). Low
temperatures (<25C) are known to discourage mating,
gonad development and spawning in P. stylirostris
(Robertson et al., 1991), P. esculentus (Crocos and Kerr,
1986), and P. semisulcatus (Aktet al., 2003). Cycling
temperature fluctuations between 20 and 28C induce
maturation and spawning in P. duorarum (Cripe, 1994)
and P. semisulcatus (Aktet al., 2003). The cycling
temperature fluctuation has been suggested to be an
effective technique in obtaining off-season reproduction in
the green tiger shrimp P. semisulcatus by the latter
authors. As a result, this study aimed also at investigating
if this technique would also be effective in inducing off-
season maturation and spawning in the pacific white
shrimp L. vannamei before this species can be suggested
for farming in sub-tropical regions in Turkey.
Therefore, this study was conducted to assess the
effects of three different induction methods (eyestalk
ablation, serotonin injection, and cycling temperature
fluctuation) in an attempt to control off-season
reproduction in L. vannamei broodstock. The main
purpose was to shift maturation and spawning towards
winter or early spring in order to enable hatcheries to
complete the whole hatchery cycle (maturation, spawning,
hatching, larval and nursery culture) before new on-
growing period begins in May, when the temperature is
warm enough for fast growth in ponds.
M. Kumlu et al. / Turk. J. Fish. Aquat. Sci. 11: 15-23 (2011)
17
Materials and Methods
Experimental Conditions
The experiment was conducted at Yumurtalık
Marine Research Station of the Faculty of Fisheries,
Adana, Turkey, for 60 days between 15 April and 15 June,
2009. The pacific white shrimp (Litopenaeus vannemei)
post-larvae (PLs) at PL8 were imported from Thailand in
the first week of July 2008 and reared in earthen ponds
until the end of October, 2008 (4 months), during which
the animals reached above 20 g. They were then over-
wintered until April, 2009 in two 3-m diameter tanks in a
green house in recirculation system before the initiation of
the experiment.
Prior to start of the experiment, all individuals were
weight to the nearest 0.01 g and shrimps (mean weight
33.5-34.5 g) were randomly allocated to each maturation
tank in five groups as described in Table 1. None of the
females stocked into the maturation tanks had gonodal
development at the beginning of the experiment. Small
maturation tanks (2 m diameter black fibreglass tanks)
connecting to a central bio-filter were preferred to
facilitate easier temperature and broodstock control for the
Groups 1-4, while a bigger tank (3 m diameter) detached
to the recirculation system was allocated to Group 5 to see
the effects of tank size and lower shrimp stocking
densities on the reproduction of the shrimps. Broodstock
stocking density was maintained as 9.44 shrimps per m2 in
Groups 1-4 and 5.67 per m2 in Group 5. A sex ratio of 1:2
(male/female) in Groups 1-4 and 1:1 in Group 5 was used
(Table 1).
The shrimps were acclimated to final experimental
conditions for a period of 7 days, water temperature in
tanks were maintained at 28°C and shrimps were fed with
fresh feeds (squid, crab and mussel) and occasionally
frozen feeds prior the start of the experiment.
After the acclimation period, the animals were
allocated to groups as follow;
Group 1 (Control): Half of female broodstock (10
shrimps) were injected with normal saline solution (8.5‰
NaCl) and the rest were remained without any treatment.
Group 2 (Hormone injected): Serotonin (5-
hydroxytryptamine, 5-HT, and creatinine sulfate complex,
Sigma, St. Louis, MO, USA) hormone injection was made
with 1 ml micro-injector at a dose of 50 µg g-1 to each
female individual from the sternite of the second
abdominal segment as in Aktaş and Kumlu (2005).
Group 3 (Eyestalk ablation): Females in this group
were unilaterally ablated by tying the eyestalk first and
then cutting it off with scissors.
Group 4 (Temperature fluctuation): Water
temperature (28°C) was lowered to 20°C at a rate of 2°C
day-1 and then kept for 2 days prior to increasing the
temperature to 28°C at the same rate. The animals were
exposed to 28°C for 2 days before the temperature was
lowered to 20°C again. This procedure was repeated in ten
days intervals, until the first ovarian development and
spawning occurred (Cripe, 1994; Aktaş et al., 2003).
Group 5 (Eyestalk ablation): The same procedure
was applied as explained before for the Group 2, except
that the stocking density and sex ratio in this group were
5.67 shrimps per m2, and 1:1 (male/female), respectively.
Each maturation tank was painted in black and had a
central outlet. In Goups 1-4, drained seawater was
recirculated through coarse filters, a submerged bio-filter
and a water heating system prior to entering into each
tank. All tanks were connected to a cooling-heating
system in order to control water temperature
independently in each tank when needed. Each tank was
fitted with 300600 W aquarium heaters as well.
Recirculation rate was adjusted to 1000% of each tank
volume per day. In addition, 5-10% fresh sea water was
supplied to recirculation system to avoid high nitrate
concentrations. Fluorescent bulbs (80 W) were hung 0.5
m above each tank to obtain the desired photoperiod (16 h
light: 8 h dark) and light intensity. Each maturation tank
was covered with a thick black polyethylene to avoid
excessive light. Light intensity on water surface was
measured with a light-meter (Model Li-250, USA) and
adjusted to <5 µE m2
s−1 as referred by Vaca and Alfaro
(2000).
Moulting, maturation and spawning of each
individual female were monitored and recorded daily. For
this propose, females were marked by placing plastic rings
of various colours around the eyestalk. Shrimps were fed
four times a day (at 09:00, 12:00, 19:00 and 24:00) until
satiation with fresh squid, mussels, and crab (Callinectes
sapidus), which were caught daily by gillnetting during
the experimental period. All the remaining wastes were
removed by siphoning the tanks before the morning
feeding.
The degree of maturation was examined externally
by inspecting the size of the developing ovaries through
Table 1. Experimental design used in the current study (Group 1: Control*, Group 2: Serotonin injected, Group 3: Eyestalk ablated,
Group 4: Cycling temperature fluctuation, Group 5**: Eyestalk-ablated)
Groups
Stocking density
(shrimp per m2)
Mean weight of
females (g)
Sex ratio
(Male/Female)
Temperature
(C)
Group 1*
9.55
34.30±3.99
1:2
28
Group 2
9.55
33.49±3.61
1:2
28
Group 3
9.55
35.25±3.40
1:2
28
Group 4
9.55
35.08±3.01
1:2
20-28 (cycles in 10 days)
Group 5**
5.67
34.60±3.09
1:1
28
* Half of the shrimps were injected with a serum solution while the other half are not treated in any way.
** This group of shrimps held in 3 m2 maturation tank, while the rest of the groups were matured in 2 m2 tanks.
18
M. Kumlu et al. / Turk. J. Fish. Aquat. Sci. 11: 15-23 (2011)
the dorsal exoskeleton on a daily basis in the evenings
(between 19.00 and 21.00 h). Each female with ripe ovaries
and carrying spermotophore was removed into a 200 L
spawning tank previously filled with filtered (down to 1
µm) and UV-irradiated seawater. EDTA (20 ppm) was
added into the spawning tanks to bind possible heavy
metals. Upon spawning, the female was returned to the
maturation tank and five 100 ml aliquot samples were
taken to determine fecundity, and the number of fertile
and infertile eggs under a microscope. The eggs were
siphoned onto a 100 µm plankton mesh and then treated
with 100 ppm formalin for 30 seconds and 50 ppm iodine
for 60 seconds before being placed into a hatching tank
(500 L) for further 36 h to determine hatching rate. An
antibiotic (furazolidone) was used in prophylactic dose
(0.5 ppm) in the hatching tanks (Anonymous, 2005;
2007). After the hatching, five 100 ml aliquot samples
were taken to count the nauplii under a microscope.
Salinity, temperature, dissolved oxygen level of each
tank were monitored daily and pH, total ammonia, nitrate,
nitrite concentrations were monitored weekly. Salinity
was measured with a refractometer, O2 and temperature
with an O2 meter (OxyGuard, Denmark) and pH with
(Thermo Orion Star 3, USA). Total ammonia, nitrate and
nitrite levels were measured with a photometer (Nova 60,
Merck).
Statistical Calculations
Data were analyzed using one-way ANOVA and
any significant difference was determined at 0.05
probability level by Scheffe`s test after normality and
homogeneity (Duncan’s test) of the data were checked in
SPSS version 17 statistical software.
Results
The initial weights for female shrimps were 34.53 ±
0.81 g while this value was 32.15± 0.75 g for males (Table
1) (P>0.05).
Throughout the experiment, water temperatures
remained between 27.5-28.5°C and 26.5-28.5°C for
Groups 1-4 and Group 5, respectively. During the
experimental period, (15 April and 15 June 2009) ambient
water temperature ranged between 18 and 22ºC. All
maturation tanks were aerated with oxygen by supplying
air continuously through air-stones from an air-blower,
thus dissolved oxygen (DO) levels remained >5 mg L-1.
pH and salinity levels of the water were measured as 8.0-
8.2 and 38-39.4‰, respectively. Ammonia (NH3-N),
nitrite (NO2−N) and nitrate (NO3-N) were 0.04-0.09, 0.05-
0.17 and 0.5-0.6 mg L-1, respectively, throughout the
study.
First ovarian maturation was observed in the
eyestalk-ablated groups (Group 3 and 5), but still the first
successful spawning occurred between the 25th and 28th
days of the experiment, in all the groups. It was observed
that females at final maturation stage mated around 19.00
21.00 h and received spermatophores. When placed
individually into spawning tanks, these females spawned
about midnight.
Throughout the experiment, a total of 9 spawning
were recorded in the control group (Group 1), 4 of which
were spawned by the females injected with saline solution
(8.5‰ NaCl) and the rest were produced by those not
treated in any way (Figure 1). 40-50% of the females
spawned during the experimental period in this group.
Mean fecundity was calculated as 48,483 for non-treated
and 52,000 eggs for saline-injected females. Mean
fertilization rate was 86-96%. Hatching rate ranged
between 16 and 31% (Table 2).
In the hormone-injected group (Group 2), a total of 8
spawning occurred and the mean fecundity was calculated
as 60,278 eggs per female. The first spawning was
recorded on the 28th day, (after the third hormone
injection) in this group (Figure 1). Fertilization and
hatching ratios were 63.08% and 18.5%, respectively
(Table 2).
In the eyestalk-ablated shrimps (Group 3), a total of
9 females spawned and mean fecundity was 79,778 eggs
per female. Eyestalk ablation stimulated ovarian
development from the first week of the experiment;
nevertheless, first successful spawning occurred on the
25th day (Figure 1). The highest fecundity per spawning
was recorded as 121,100 eggs in this group. Mean
fecundity ratio was above 88%, however, hatching rate
remained at just 8.53% (Table 2). In this group, 55% of
the females spawned during the experimental period.
In Group 4 (temperature fluctuation), 7 females
spawned producing an average of 28.500 eggs per female.
Ovarian development was stimulated after the third
temperature fluctuation and the first spawning occurred on
the 25th day (Figure 1). The highest number of fecundity
per female was recorded as 62,000 eggs. Fertilization and
Table 2. Number of spawns, spawning rate, fecundity, fertilization rate, hatching rate of the females during the experiment
Groups
Number of
spawns
Spawning rate
(% female)
Mean fecundity
(per female/spawn)
Mean fertilization
rate (%)
Mean hatching
rate (%)
Group 1
5
50.00
48,483±12,980bc
86.25±11.25a
31.00±7.12a
4*
40.00
52,000±16,513bc
95.75±5.06a
16.22±6.73b
Group 2
6
35.29
60,278±17,745bc
63.08± 13.23b
18.50±3.81b
Group 3
11
55.00
79,778±19,933b
88.37±8.02a
8.53±2.15c
Group 4
7
38.89
28,500±18,624d
86.54±12.57a
10.13±1.83c
Group 5
18
90.00
125,015±29,276a
79.06±13.21ab
28.55±7.06a
In Group 1, * indicates the number of saline solution-injected females, while the rest are non-treated females. Each value for fecundity, fertilization and
spawning rate is a mean ± standart deviation. Means marked with different letters at the same column are significantly different from each other (P<0.05).
M. Kumlu et al. / Turk. J. Fish. Aquat. Sci. 11: 15-23 (2011)
19
hatching rates were 86% and 10%, respectively (Table 2).
%39 of females in this group spawned during the
experimental period.
In the second eyestalk-ablated group (Group 5), a
total of 18 females (%90 female spawning rate) spawned
generating the highest mean fecundity of 125,015 eggs per
female (P<0.05). Eyestalk ablation stimulated ovarian
development from the first week of the experiment; but
the first successful spawning occurred on the 25th day
(Figure 1). The highest fecundity (219,000 eggs per
female) was recorded in this group (P<0.05). Fertilization
and hatching rates were 79% and 28.55%, respectively
(Table 2). The highest hatching rate was determined as
32.24%.
At overall, the highest (125,015 eggs) and the lowest
(28,500 eggs) average fecundity were observed in Group
5 and Group 4 (P<0.05), respectively, during the
experimental period (Table 2). More females spawned and
had better overall reproductive performance in Group 5
compared to other treatments. Fertilization rate (63.08%)
Figure 1. Fecundity (eggs per female) and spawning time of the individual shrimps over the entire experimental periods.
Group 1: Control, Group 2: Serotonin injected, Group 3: Eyestalk-ablated, Group 4: Cycling temperature fluctuation, Group 5: Eyestalk-ablated
In Group 1, * indicate the fecundity values of saline solution-injected individuals, while the rest are non-treated females.
20
M. Kumlu et al. / Turk. J. Fish. Aquat. Sci. 11: 15-23 (2011)
was found to be significantly lower in the serotonin-
injected group (Group 2) among all the treatments
(P<0.05). In general, hatching rates ranged between 9%
and 30%, and were lower than expected. The highest
hatching rates were observed in the control (Group 1; in
non-treated shrimps) and Group 5 (29-31%) and the
lowest were found in Group 3 and Group 4 (P<0.05).
Throughout the experimental period, broodstock survival
rate remained above 90%.
In the microscopic examinations, even in the best
spawnings, morphological apperances of some of the eggs
were observed to be abnormal (ellipsoid shapes, extremely
large or small in size, punctured membranes etc.). These
abnormalities continued during the course of experiment.
A part of eggs, showing normal embryonic development,
hatched out successfully and the larvae could be cultured
up to post-larvae.
Discussion
Eyestalk ablation is still the most effective and
common method used for the induction of ovarian
maturation in penaeid shrimps. As with other species
(Browdy and Samocha, 1985; Bray and Lawrence, 1992;
Browdy, 1992; Aktaş et al., 2003), the eyestalk ablation
was found to be the best technique in the off-season
maturation and spawning of the Pacific white shrimp L.
vannamei in our study. Despite being in off reproduction
season, 55-90% of the eyestalk- ablated females were able
to successfully develop their ovarium and spawn within
the 2-months experimental period. The shrimps in Group
3 (eyestalk-ablated) displayed the best performance in
terms of the rate of spawned females and fecundity
compared to the other groups which matured at the same
tank size and stocking density (Groups 1, 2, and 3).
Throughout the experiment, 90% of the females in Group
5 spawned and produced an average of 125,000 eggs per
female with 79% fertilization rate. In agreement with Bray
and Lawrence (1992), Browdy (1992), and Aktaş and
Kumlu (1999), the eyestalk-ablation generated more
spawnings and egg-production, but not higher fertilization
or hatching rates in our study.
Size of maturation tanks and broodstock stocking
density are known to influence matings and ovarian
development in shrimps (Primavera, 1979; Crocos and
Kerr, 1986). In our study, a significant difference in the
reproductive performance found between the two
eyestalk-ablated groups (Group 3 and 5) might be due to
tank size and/or stocking density. In fact, these factors are
known to have different effects on reproductive
performance of shrimps. For example, in a study we
carried out with the green tiger shrimp (P. semisulcatus),
we had good results in 1.2 m diameter tanks at 1:2
male/female ratio and 10 shrimps per m2 (Aktaş et al.,
2003). In this study, we found similar reproductive
performances (spawning rate, fecundity, fertilization and
hatching rates) in this species in either small (1.2-m) or
large (4-m) broodstock tanks. In the current study with L.
vannamei, however, the females in 3-m tank at 5.67
shrimps per m2 (Group 5) produced significantly (>36-
60%) more eggs per female and spawning rate (90%)
compared to those held in 2-m tanks at 9.55 shrimps per
m2 in the Group 1 (control) or Group 3 (eyestalk-ablation).
Yet, tank size, sex ratio, and/or stocking density did not
have any positive effects on either fertilization or hatching
rate in the present study. Based on our results and those at
the literature (Browdy and Samocha, 1985; Crocos ve
Kerr, 1986), it can be concluded that broodstock tanks of
not smaller than 3 m in diameter have to be preferred for
the successful reproductive performance of L. vannamei.
Chen et al. (1991) suggested the use of at least 6 m2 of
tank bottom for L. vannamei broodstock.
Our broodstock size (30-35 g) is similar to what
Yano (1993) suggested to be optimum (25-35 g) for L.
vannamei. A sex ratio of 1:1 (male/female) is very
common, but some hatcheries prefer 1:1.5-2.5
(male/female) ratio for this species. Stocking density can
be between 5 and 10 animals per m2 (Chamberlain and
Lawrence, 1985; Treece, 1999). In a recirculation system,
Chen et al. (1991) obtained good results with 10 shrimps
per m2 stocking density, which we also used in Group 5 in
our study.
In general, long photoperiods and temperatures
above 25C are known to be suitable for maturation of
many shrimp species such as Penaeus japonicus (Laubier-
Bonichon, 1978), P. setiferus (Brown et al., 1979), P.
esculentus (Crocos and Kerr, 1986), P. duorarum (Cripe,
1994) and P. semisulcatus (Aktaş et al., 2003). In sub-
tropical regions, temperatures <25C, encountered during
late-autumn, winter or early-spring, depress gonad
development and spawning in shrimps (even in eyestalk-
ablated females) (Crocos and Kerr, 1986; Robertson et al.,
1991; Aktaş et al., 2003). Despite providing optimal
conditions, shrimp broodstock may not readily develop
ovaries and spawn in off-reproductive season in captivity,
but applying cyclic temperature fluctuations between
optimal and sub-optimal levels (20 and 28C) have proven
to induce maturation and successful spawnings in P.
duorarum by Cripe (1994), and in P. semisulcatus by
Aktaş et al. (2003). The latter researchers obtained similar
results with this technique as eyestalk-ablation even in
winter months (off-season) in P. semisulcatus. However,
although cyclic temperature fluctuations appeared to
induce maturation and spawning after the third cycle in L.
vannamei, the effect on reproductive performance was
very poor (P<0.05). Therefore, L. vannamei seems to be
responding differently to this treatment than the other
shrimp species, and that the cycling temperature
fluctuation technique, as we used in this study, is not
recommended for off-season maturation and spawning of
this shrimp species.
Several studies have shown some positive effects of
hormone injections on reproduction of decapod
crustaceans (Vaca and Alfaro, 2000; Alfaro et al., 2004;
Aktaş and Kumlu, 2005; Ngernsoungnern et al., 2008;
Santoshi et al., 2009), but there is no study dealing with
the influence of hormones on off-season maturation in
penaeid shrimps. Although serotonin was found to induce
maturation and spawning in L. vannamei (Vaca and
Alfaro, 2000) and P. semisulcatus (Aktaş and Kumlu,
M. Kumlu et al. / Turk. J. Fish. Aquat. Sci. 11: 15-23 (2011)
21
2005), this hormone poorly induced maturation and
spawning in L. vannamei in the current study. The reason
for this might be due to some unknown seasonal response
(winter conditions) of this shrimp species to hormone.
Time between induction and first spawnings in off-
season studies depends on the treatments and may change
from one shrimp species to another. Aktaş et al. (2003)
obtained the first spawnings on the 13th and 33rd days in
eyestalk-ablated and temperature-fluctuated groups,
respectively, in P. semisulcatus. Crocos and Kerr, (1986)
also reported similar response for P. esculentus. In the
cyclic temperature fluctuated group, Cripe (1994)
obtained the first spawnings on the 20-25th days of the
experiment in P. duorarum. In our study with L.
vannamei, despite much earlier ovarian development (first
week) we observed in the eyestalk-ablated groups, the first
spawnings occurred between the 25th and 28th days of the
experiment, irrespective of the maturation treatments.
Throughout the experiment, some of the spawnings
resulted in poor egg quality regardless of the experimental
groups. Even in the best spawnings, morphological
apperances of some of the eggs were observed to be
abnormal (ellipsoid shapes, large or small sizes, punctured
membranes etc.). In general, fertility rates were high but
hatching rates were unexpectedly low. Many factors such
as low water quality, inappropriate photoperiod,
insufficient quantity or quality of the feeds or even
genotype of the broodstock migh account for low hatching
rates (Menasveta et al., 1994). It is well-known that
nutrition is one of the main factors influencing gonad
development in shrimps. In commercial hatcheries,
broodstock are generally fed on fresh seafoods (mussel,
oyster, squid, crab or sea worms) and sometimes artificial
feeds until satiation for successful maturation and
spawnings (Primavera, 1978; Chamberlain, 1985;
Makinouchi and Primavera, 1987; Palacios and Racotta,
2003). Similarly, in our study, we also fed the broodstock
ad libitum on fresh and occasionally on frozen squid, crab
and mussel to provide nutritionally balanced diet. In our
earlier studies, with similar feeding regimes we obtained
good reproductive performances in P. semisulcatus, even
in off-season periods (Aktaş and Kumlu, 1999; Aktaş et
al., 2003).
In addition to water temperature, photoperiod, light
intensity and quality, many other water quality parameters
(i.e. salinity, pH, dissolved oxygen, nitrogenous wastes,
heavy metals etc.) might also influence spawning success
in penaeid shrimps (Primavera, 1985; Harrison, 1990). In
order to ensure best water quality, we recirculated the
seawater through sand filters, 1 µm cartridge filters , and a
UV system at least 5-6 times prior to using in the
spawning and hatching tanks. EDTA, an antibiotic
(furazolidone) and povidone iodine were also used to
chelate heavy metals and control infections (Anonymous,
2005; 2007). As a result, we never observed any kind of
disease throughout the experiment. The photoperiod,
temperature, salinity, pH and all other environmental
parameters were adequate for good hatchery practices.
The stress encountered in captivity is know to exert
negative impacts on reproductive performance and gamet
quality in both males and females of shrimps (Chen et al.,
1991; McVey, 1983). In addition to the stress that the
broostock are facing in captivity, forcing them to
reproduce out of the season might have also exacerbated
the problem. In a study we carried out with P.
semisulcatus in winter months, despite maintaining
adequate maturation and spawning conditions, we also
faced with problems of low hatching rates, which we were
unable to rectify during the entire study (Aktaş et al.,
2003).
In general, the use of pond-reared broodstock in
hatcheries results in lower reproductive performance
compared to wild counterparts. Benzie (1997) reported
serious larval quality problems from the domesticated
broodstock of P. japonicus or L. vannamei, both of which
are considered to be relatively easy shrimp species to
reproduce in captivity. It is also know that inbreeding
depressions might often be seen when pond-reared
animals are used as broodstock for several generations
(Sbordoni et al., 1986). When a new species is imported
into a country and its life-cycle is closed, the reproductive
performance might seriously be hampered due to low
genetic variation (Goyard et al., 2003). In fact, in the
breeding programs with low genetic variation, inbreeding
depressions migh occur even after 1 to 2 generations.
Moss et al. (2008) found 33.1-47.1% lower hatching rates
in L. vannamei due to inbreeding depressions even after
two generations. These researchers reported that the stress
exerted by environmental parameters worsen the situation
further. As a result, the low hatching rates we obtained in
the current study migh have been influenced by the stress
due to captive conditions, season, as well as genotype of
the broodstock of L. vannamei.
Recently, faster growing and disease-resistant strains
(SPR: Specific Pathogen Resistant of SPF: Specific
Pathogen Free) through breeding programs have been
developed in many countries (Cuzon et al., 2004). Some
commercial companies are known to produce and sell off
their post-larvae from genetically modified broodstock,
and when the purchased seeds are grown in ponds and re-
used again as broodstock by the farmers, immediate
inbreeding deformations occur. Therefore, if L. vannamei
is to be produced in the sub-tropical regions in Turkey, we
have to either bring the fast growing SPF or SPR post-
larvae for only on-growing purposes from abroad, or
establish our own selective breeding program for this
shrimp species.
The results of this study has demonstrated that,
under Mediterranean climatic conditions, the broodstock
of this non-indigenous shrimp species can be readily
matured and spawned out of season in recirculating
systems, but further research has to be carried out to
improve hatching rate.
Acknowledgements
This study was funded by Scientific and Technical
Research Council of Turkey (TUBITAK) with Project
Number of 1009O004.
22
M. Kumlu et al. / Turk. J. Fish. Aquat. Sci. 11: 15-23 (2011)
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... Puede reproducirse rápidamente en cautiverio, posee un amplio rango de tolerancia a factores ambientales como la temperatura y la salinidad, tiene una buena asimilación en dietas que contienen bajos porcentajes de proteína y crecen rápido en comparación con otras especies de camarones peneidos (Kumlu et al., 2011). Sus rangos de tolerancia tanto de temperatura como de salinidad, van de los 15°C a los 33°C y de 0.5 a 40 psu, respectivamente (Briggs et al., 2004;FAO, 2013;Ramos-Cruz, 2011;Re et al., 2012). ...
... Por lo tanto, debido al importante papel que juega el pedúnculo ocular en Litopenaeus vannamei no sólo en la reproducción, sino también en la respuesta a nivel molecular del organismo ante estas condiciones externas, esta investigación pretende evaluar la respuesta de Litopenaeus vannamei a la exposición a la temperatura crítica máxima en organismos ablacionados unilateral y bilateralmente, y aclimatados a diferentes temperaturas mediante la expresión de genes en diferentes vías metabólicas y de algunas proteínas involucradas en la protección y mantenimiento celular. Estas especies pueden reproducirse rápidamente en cautiverio y en crecerse a altas densidades (por encima de 400 m 2 en sistemas de recirculación), tienen un amplio rango de tolerancia a parámetros ambientales como la temperatura y salinidad, asimilan mejor dietas que contienen bajos porcentajes de proteína, poseen una alta resistencia a enfermedades, cuentan con tasas altas de sobrevivencia larval (50-60%) y tienen una tasa de crecimiento relativamente rápida (más de 3g por semana) en comparación con otras especies de camarones peneidos (Re et al., 2012a;Kumlu et al., 2011). Es una especie eurihalina capaz de tolerar salinidades en el rango de 1 a 40 psu, característica ecológica que lo hace un candidato idóneo para su cultivo en ambientes controlados (FAO, 2013;Ramos-Cruz, 2011). ...
... Este procedimiento conduce a picos predecibles de maduración y desove que facilitan el establecimiento de programas de producción, en contraste con desoves dispersos de hembras no sometidas a la EA. Varios estudios han demostrado la ventaja de este procedimiento debido al incremento en la producción de una mayor cantidad de desoves, ovocitos y nauplios totales por hembra (Kumlu et al., 2011;Palacios et al., 1999). Las hembras ablacionadas adquieren una maduración más rápida, pero no altas tasas de fertilización y eclosión (Kumlu et al., 2011). ...
Thesis
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Litopenaeus vannamei es una especie económicamente importante y su cultivo se ha extendido a nivel mundial. Para acelerar las tasas de reproducción se han implementado técnicas como la ablación de pedúnculo ocular para inducir la maduración gonadal en criaderos comerciales de camarones peneidos. El pedúnculo ocular contiene un complejo sistema neuroendocrino, el cual involucra elementos reguladores que le permiten responder a factores externos como la temperatura, salinidad, estrés, entre otros, mediante el ajuste de sus funciones vitales. En este estudio se evaluó el efecto de la ablación en branquia de Litopenaeus vannamei ablacionados y no ablacionados aclimatados a 20, 26 y 32°C, los cuales fueron sometidos a estrés térmico agudo a través de un análisis trancriptómico de genes involucrados en procesos como la regulación iónica y permeabilidad membranal, hipoxia y estrés térmico. Se obtuvieron valores de temperatura crítica máxima en un rango de 33.3-41.4°C. Mediante la validación de genes de referencia realizada se utilizaron los genes L8 y Ef1α para la normalización de los genes blanco (Hsp70, Hsp90, Hif1α y Na+/K+ATPasa). De acuerdo con los valores obtenidos de expresión relativa normalizada se encontró que la ablación induce la expresión del gen Hsp70 en organismos sometidos a estrés térmico agudo, independientemente de la temperatura de aclimatación. El gen HSP90 mantuvo niveles bajos y estables de expresión bajo las temperaturas de aclimatación de 20 y 26°C en organismos sometidos a estrés térmico agudo. Sin embargo, en organismos experimentales aclimatados a 32°C se obtuvo una expresión significativa de este gen y al adicionar el efecto de la ablación, la expresión se vio sobre expresada a niveles más altos. El gen Hif1α mostró una mayor expresión en organismos control que en organismos experimentales. Y para el caso del gen Na+/K+ ATPasa se observó una sobre expresión del gen, aunque no se presentaron diferencias significativas en la expresión del mismo. De acuerdo con los niveles de expresión de los genes Hif1α y Na+/K+ ATPasa se encontró que posiblemente existe un efecto de corregulación entre estos genes debido a la función que desempeñan. En general la regulación de la expresión de los genes evaluados en este trabajo, se vio afectada en los organismos ablacionados, lo cual indica que la eliminación de parte del sistema neuroendocrino de L. vannamei debido a la ablación, causa un desbalance energético y una des-regulación de los genes en los organismos expuestos a estrés térmico, los cuales se encuentran participando en procesos de osmoregulación, hipoxia y estrés térmico.
... Specifically, recent literature has found that Pacific White Shrimp ( Litopenaeus vannamei), the most consumed species of shrimp in the United States, can be bred in the off-season and in subtropical conditions. This development would enable farmers to breed their own shrimp as opposed to importing them from coastal hatcheries [5]. Due to the low salinity sensitivity of Pacific White Shrimp, this discovery suggests the possibility of developing self-sustaining Pacific White Shrimp farms in unconventional regions far from coastal regions and natural breeding grounds. ...
... Thus, shrimp spend about 610 days of their life with the ability to reproduce. Furthermore, shrimp reproduce around three times over their lifespan, and produce approximately 2,240 offspring each time [5]. Thus, the likelihood that any given shrimp is reproducing on any given day is 3/610, or 0.004918. ...
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... Record the number of stage 4 females which are transferred to the mating tank (male tank). During the mating period (3-10 pm), turn on the lights over the mating tanks [17]. ...
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... The manipulation of stocking density is an important consideration for optimizing growth and survival during culture of many crustaceans. High stocking densities have been shown to affect biochemical composition , growth (Naranjo-P aramo et al. 2004), reproduction (Kumlu, Turkmen & Eroldogan 2011) and moult increment of crustaceans. Moult increment and moult cycle duration are genetically set at optimum levels, but are then modified by nutrition and other factors (Freeman 1990) such as photoperiod and stocking density. ...
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... It is similar to the spawning temperature for P. semisulcatus at 24-28°C in Turkey (Kumlu et al., 1999). The rise in temperature not only triggers the gonad development of M. palmensis, but may also be associated with the cycle and success of the molting stage (Yamada et al., 2007;Kumlu et al., 2011), as well as limiting the northern boundary of their geographical distribution (Sakamoto & Hayashi, 1977). ...
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