Inducing synchronous ovarian maturation in the

craysh, Procambarus clarkii, via eyestalk

interventional injection as compared with eyestalk
ablation and combined injection of serotonin and
domperidone
Shengli Liu
1,2
, Shiyuan Gong
1,2
, Jinmei Li
2
& Wenhu Huang
2
1
Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education, Huazhong
Agricultural University, Wuhan, China
2
College of Fishery, Huazhong Agricultural University, Wuhan, China
Correspondence: S Liu, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education,
Huazhong Agricultural University, Wuhan 430070, China. E-mail: victoryliu@yahoo.cn
Abstract
We examined the feasibility of inducing synchro-
nous ovarian maturation in the craysh, Procamb-
arus clarkii, via eyestalk interventional injection
(EI) using a Bletilla striata polysaccharide (BSP)
gelatin containing tranexamic acid (TRA), dom-
peridone (DOM) or serotonin (5-HT). In total, 360
females were randomly assigned to seven experi-
mental groups and a control group. The average
survival rate (SR%) and average synchronous
ovarian maturation rate (SMR%) in survivors in
the EI groups, EI-TRA, EI-DOM, EI-5HT were
compared with bilateral eyestalk ablation (BEA),
unilateral eyestalk ablation (UEA), abdominal
injection (AI) of DOM (0.5 mg craysh
1
) alone
(AI-DOM) or combined with 5-HT (0.5 mg cray-
sh
1
) (AI-DOM+5-HT). The experiment covered a
prolonged period of 32 days until two ovigerous
females were observed in BEA. EI-DOM achieved a
SMR (66.67 9.62%) higher than the control
(22.05 3.06%) (P < 0.05), but lower than BEA
(88.89 11.11%), UEA (63.33 18.56) and
AI-DOM+5-HT (59.26 10.14%) (P > 0.05).
EI-DOM also achieved a higher SR (53.33 3.85%)
than BEA (15.56 2.22), UEA (24.44 5.88)
(P < 0.05) and AI-DOM+5HT (51.11 4.44)
(P > 0.05). However, EI-TRA (SR = 28.89 4.44,
SMR = 37.78 2.22) and EI-5-HT (SR = 15.56
4.44, SMR = 22.22 11.11) failed to induce
signicantly higher SMR than the control. These
ndings suggest that EI methods, such as EI-DOM,
may have positive characteristics for the develop-
ment of inexpensive and less labour-intensive
techniques for induction of ovarian maturation in
decapods.
Keywords: Bletilla striata polysaccharide, Eyestalk
interventional injection, Ovarian maturation,
Procambarus clarkii
Introduction
Currently, at least two methods have been proved to
be effective in accelerating ovarian maturation of
economic decapod crustaceans. One method is eye-
stalk ablation, which was considered to be one of the
determinant factors for mass production of quality
stocking seeds in captive penaeid shrimps (Santiago
1977; Primavera 1978; Choy 1987), and also in spe-
cies such as crab Potamon persicum (Khazraeenia &
Khazraiinia 2009), craysh Cherax quadricarinatus
(Sagi, Shoukrun, Levy, Barki, Hulata & Karplus
1997), as well as prawn Macrobrachium (Okumura &
Aida 2001; Varalakshmi & Reddy 2010). The other
method is injecting with serotonin (5-HT) and/or a
dopamine (DA) antagonist. Injection with 5-HT
alone was reported to induce ovarian maturation in
red swamp craysh Procambarus clarkii (Sarojini, Na-
gabhushanam & Fingerman 1995b), white shrimp,
Litopenaeus vannamei (Vaca & Alfaro 2000), black
tiger shrimp, penaeus monodon (Wongprasert,
2012 John Wiley & Sons Ltd 1402
Aquaculture Research 2014, 45, 14021414 doi: 10.1111/are.12086
Asuvapongpatana, Poltana, Tiensuwan & With-
yachumnarnkul 2006), indian white prawn,
Fenneropenaeus indicus (Santhoshi, Sugumar & Mun-
uswamy 2009) and banana shrimp, Fenneropenaeus
merguiensis (Zacharia & Kakati 2011). Injecting 5-HT
(2.5 9 10
7
mol or 2.5 9 10
6
mol per prawn)
into prawns of M. rosenbergii signicantly increased
vitellogenin (Vg) concentration in the haemolymph
at ovarian stage IV compared with control groups
(Tinikul, Mercier, Soonklang & Sobhon 2008).
Injection with a DA antagonist, SCH23390
(1.25 9 10
5
mol per prawn) suppressed DAs effect
on vitellogenin synthesis and increased haemolymph
Vg concentrations of M. rosenbergii (Chen, Fan, Hsieh
& Kuo 2003). A combined injection with 5-HT
(25 lg g
1
b.w.) and a DA antagonist, spiperone
(1.5 or 5 lg g
1
b.w) induced ovarian maturation
and spawning in wild Litopenaeus stylirostris and
pond-grown L. vannamei (Alfaro, Z u~ niga & Komen
2004). Injection with 5-HT and/or DA antagonists
remains unsatisfactory because it is expensive and
must be performed periodically, while eyestalk
removal frequently kills the females either at the time
of surgery or after the operation, and leads to perma-
nent damage.
The successful articial propagation of any crus-
tacean requires an understanding of the endocrine
mechanism and control of gamete maturation.
The commonly accepted theory explaining ovarian
maturation induced by eyestalk ablation proposes
that there is an important X organ (XO) and a
connected sinus gland (SG) in the eyestalk. This
XO-SG complex is viewed as a unique endocrine
organ in decapod crustaceans, which prevents pre-
cocious maturation of the ovary in non-breeding
seasons (Bray & Lawrence 1992; Fingerman
1995). Nagaraju (2011) reported that at least two
types of peptides synthesized and stored in the
XO-SG were responsible for this effect on ovarian
maturation. One of these peptides is the gonad-
inhibiting hormone (GIH), which antagonizes the
gonad-stimulating hormone (GSH), a hormone
believed to be synthesized in the brain and
thoracic ganglia (TG) rather than eyestalk tissue
(Fingerman 1995; Yano, Tsukimura, Sweeney &
Wyban 1988; Yano 1992). The other is a mandib-
ular organ-inhibiting hormone (MOIH) proposed
by Chaves (2001) and Li, Wagner, Friesen and
Borst (2003), which inhibits the mandibular organ
(MO) from synthesizing and secreting a methyl
farnesoate (MF). The MF is an unepoxidated form
of insect juvenile hormone (JH) III, which is highly
suspected to be an important reproductive hor-
mone in decapod crustaceans (Nagaraju, Reddy &
Reddy 2006; Nagaraju 2007; Sagi, Ahl, Danaee &
Laufer 1994). However, it remains unclear
whether embolizing the eyestalk with a non-
dispersible haemolymph coagulant would accelerate
ovarian maturation.
The roles of DA and 5-HT in the reproduction of
decapod crustaceans have also been studied exten-
sively. In M. rosenbergii and L. vannamei, the
changes in DA and 5-HT levels in the central ner-
vous system (CNS) and ovary during the ovarian
cycle (Tinikul et al. 2008; Tinikul, Poljaroen,
Kornthong, Chotwiwatthanakun, Anuracpreeda,
Poomtong, Hanna & Sobhon 2011), and the
changes in haemolymph Vg concentrations caused
by 5-HT injection (Tinikul et al. 2008) or DA
injection (Chen et al. 2003) supported the
judgment that 5-HT stimulates ovarian develop-
ment (Kulkarni, Nagabhushanam, Amaldoss,
Jaiswal & Fingerman 1991b, 1992; Meeratana,
Withyachumnarnkul, Damrongphol, Wongprasert,
Suseangtham & Sobhon 2006), while DA inhibits
it (Sarojini, Nagabhushanam & Fingerman 1996;
Sarojini, Nagabhushanam, Devi & Fingerman
1995a). In P. clarkii, the modes of action of 5-HT
in stimulating ovarian maturation and DA in
inhibiting accelerating ovarian maturation, which
may be caused by 5-HT injections, were explained
by 5-HT stimulating the release of a GSH in the
brain and thoracic ganglia (Sarojini et al. 1995b),
while DA may inhibit GSH release in these tissues,
or stimulate GIH release from the eyestalks, or
both (Chen et al. 2003; Fingerman 1997; Sarojini
et al. 1995a). DA is a signicant antagonist of
5-HT and is thought to be channelled through the
D1 rather than the D2 receptors of DA to exert its
inhibitory effects on ovarian maturation (Chen
et al. 2003). The D2-type antagonist, domperi-
done, showed no signicant effect on ovarian mat-
uration induction, while the D1-type antagonist,
SCH23390 (Chen et al. 2003) or spiperone (Alfaro
et al. 2004) induced ovarian maturation. Hence,
the widely used ovarian induction agent, domperi-
done, is of no use in decapod breeding.
As the XO-SG located in the eyestalk has been
postulated to control gonad development in crusta-
ceans, the eyestalk ganglia were also examined to
determine the localization and release of 5-HT
(Rodriguez-Sosa, Picones, Rosete & ArEChigaY
1997) and DA (Alvarez, Villalobos, Rosete, Sosa &
Arechiga 2005). In the eyestalks of P. clarkii, the
2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 14021414 1403
Aquaculture Research, 2014, 45, 14021414 Eyestalk intervention for maturation induction S Liu et al.
basal level of 5-HT detected by Rodriguez-Sosa
et al. (1997) was 95.4 49.3 pg mg
1
wet mass,
which was comparable to the value reported by
Kulkarni et al. (1992) of 102 pg mg
1
wet mass.
5-HT was present in all four ganglia of the eye-
stalk and the highest proportion (40.2%) was
found in the medulla terminalis (MT) (Rodriguez-
Sosa et al. 1997). The DA content in the eyestalks
of P. clarkii was reported by Alvarez et al. (2005)
to be 5.6 0.1 pmol (about 1062 18.9 pg) per
structure; the highest content was also found in
the MT (over 60%). Using electrical stimulation
and high concentrations of K
+
and Ca
2+
,
enhanced levels of DA and 5-HT were also
detected (Alvarez et al. 2005; Rodriguez-Sosa et al.
1997). These studies suggested that eyestalks
might play an important role in the release of DA
and 5-HT to control ovarian development.
However, the safety and effect of eyestalk interven-
tional injection with a DA antagonist or 5-HT
have not yet been documented in vivo. In addition,
an eyestalk intervention method has not previ-
ously been reported.
In general, an interventional injection requires
gelatin as an agent carrier. The gelatin should
be a non-dispersible, sustained-releasing and
harmless material. The polysaccharide extracted
from Bletilla striata (BSP) appears to be a non-
dispersible and sustained-releasing bioadhesive
material and has been used as an embolizing
agent (Chuansheng, Gansheng & Huimin 1998;
Feng, Kramann, Zheng & Zhou 1996; Zheng,
Feng & Zhou 1996) and a non-viral gene vec-
tor (Xiang-wen, Xin, Gan-sheng, Yan-bing &
Chuan-sheng 2008) in recent years. This polysac-
charide is the effective constituent of a Chinese
Herbal Medicine, B. striata (Chinese name is Bai
Ji) and is used to stop bleeding caused by trau-
matic injuries, heal wounds, reduce swelling,
and promote regeneration of tissue (Yeung
1985). BSP does not inuence the endocrine reg-
ulation of decapods and can prevent excessive
bleeding after injections when is used as a car-
rier of eyestalk interventional injection agents.
The aim of our study was to examine the feasi-
bility of inducing synchronous ovarian maturation
in red swamp craysh, P. clarkii, via eyestalk inter-
ventional injection with non-dispersible haemol-
ymph coagulant, or sustained-release 5-HT or
DOM. We used BSP as a carrier for tranexamic
acid (TRA, used as a haemolymph coagulant),
5-HT or DOM when injected into the eyestalks of
P. clarkii. The D2-type DA antagonist, domperi-
done, was also evaluated in ovarian maturation
induction of P. clarkii when injected alone or com-
bined with 5-HT.
Material and methods
Experimental animals
Collection of P. clarkii
Mature female P. clarkii (2530 g body weight
and 7080 mm measured from the base of the
eyestalk to the end of the telson) were obtained
from an aqua farm in Wuhan city, China in late
August, just before the peak season of their ovar-
ian maturation in this area (Gong, Lv, Sun, Li &
He 2008), of 2009 and 2010. The craysh had
thicker carapace with yellow granules. After selec-
tion, a random sample of 30 females from 925
female craysh showed that all were in ovarian
stage III to ovarian stage IV (ovarian stage register
see section Sample processes and ovarian develop-
ment stages register).
Maintenance of P. clarkii
All selected craysh were immediately transferred
into the laboratory after collection. First, they were
placed into a plastic tank with a water depth of
1.5 cm and left for 4 h. The active ones were then
selected and maintained in 120 9 60 9 50 cm
self-circulation aquariums with a temperature reg-
ulating device under a photoperiod 12L:12D. Cray-
sh were given surimi (70% in total food weight)
and fresh vegetable (30% in total food weight) and
fed ad libitum. Every aquarium had 35 cm of
water, 15 articial caves ( = 5 cm, L = 18 cm),
2 polyethylene climbing nets set on a substratum
of gravel and was given constant aeration. The
density of craysh was 15 females per aquarium.
To acclimatize them to lab conditions, all craysh
were cultured for 8 days at approximately
20~26C before treatment. During acclimatization,
dead individuals were quietly replaced by active
ones during the 8 days.
Agent preparation
Solutions
(1) A craysh saline solution (Vans solution):
205 mM Na
+
, 5.4 mM K
+
, 13.5 mM Ca
2+
,
2.5 mM Mg
2+
, 241.4 mM Cl

, 2.5 mM Hepes
(Van Harreveld, 1936).
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Eyestalk intervention for maturation induction S Liu et al. Aquaculture Research, 2014, 45, 14021414
(2) A 0.1 g mL
1
TRA solution: tranexamic acid
(TRA) (Alfa-Aesar, CAS: 1197-18-8) was dis-
solved in the Vans solution and made into a
0.1 g mL
1
solution.
(3) A 2 mg mL
1
DOM solution: 200 mg dom-
peridone (Sigma-Aldrich, CAS: 57808-66-9)
was dissolved in 2 mL 99.9% (v/v) alcohol
and well ground in a glass tissue homoge-
nizer, then adjust to 100 mL with Vans solu-
tion to make a 2 mg mL
1
DOM solution.
(4) A 2 mg mL
1
5-HT solution: serotonin hypo-
chloride (Sigma-Aldrich, CAS: 153-98-0) was
dissolved in the Vans solution and made into
a 2 mg mL
1
solution.
Interventional injection agent
BSP extract. The pulverized powder of the dried
root of B. striata was purchased from the Tongren
Drugstore in Wuhan, China. To extract BSP, we used
a modied form of the alcohol-precipitation method
used by Xiang-wen et al. (2008). Our extraction
steps included cold infusion (V
water
/M
B. striata
= 15/1)
at 4C for 1824 h, a water bath at approximately
70C for 1.52 h, centrifugation (1400 g, 30 min)
using a Hettich centrifuge (ROTO SILENTA R/RS),
graded alcohol-precipitation using 85% (v/v) and
95% (v/v) alcohol, ltration using a copper screen
(sieve mesh number was 500), dehydration three
times using 99.9% (v/v) alcohol, sifting using a silk
gauze (sieve mesh number was 300), and then blow
drying using warm air. The dry powder of BSP was
kept in a refrigerator at 4C until the intervention
agent was prepared.
Preparation of the gelatin containing TRA, 5-HT or
DOM using BSP. BSP (6 g) was dissolved in
100 mL of 0.1 g mL
1
TRA solution, 2 mg mL
1
DOM solution or 2 mg mL
1
5-HT solution to
obtain a gelatin containing TRA, 5-HT or DOM for
eyestalk interventional injection. Our steps
included soaking BSP in 0.1 g mL
1
TRA solution,
2 mg mL
1
DOM solution or 2 mg mL
1
5-HT
solution at 4C for 4 h, adjusting to 100 mL with
the same solution, mixing using ultrasonic waves
(for 5 s with an interval time of 10 s [Sonics,
JY92-II]) in an ice bath and addition of the same
solution until the volume reached 100 mL, suc-
tion ltration through a copper screen sieve (mesh
number was 500), mixing again using ultrasonic
waves (as before), and then conrming that the
gelatin could pass through a 30-G needle.
Experimental design
After acclimatization, 30 P. clarkii individuals were
sacriced to conrm their ovarian development
stages (only 3 of the 30 individuals had reached
ovarian stage IV), and 360 females were randomly
assigned to 24 aquariums (15 females per aquar-
ium). The aquariums were divided into eight
groups (7 experimental groups and one control
group) as follows: Craysh in the seven experimen-
tal groups were treated with bilateral eyestalk
ablation (BEA); unilateral eyestalk ablation (UEA);
eyestalk intervention with a gelatin containing
0.1 g mL
1
TRA (EI-TRA), 2 mg mL
1
DOM
(EI-DOM) or 2 mg mL
1
5-HT (EI-5-HT); abdomi-
nal injection at 3-day intervals with 2 mg mL
1
domperidone and 2 mg mL
1
5-HT (AI-DOM+5-
HT), or 2 mg mL
1
domperidone (AI-DOM) only.
The control group was untreated.
When two ovigerous females were observed in
BEA, which took a prolonged period of 32 days,
all the females in the eight groups were dissected.
The average survival rate (100N
survived
/15 = SR%)
and ovarian synchronous maturation rate of the
survivors (100N
ovary-matured
/(15-N
died
)=SMR%) in
the three parallel aquariums in each group as well
as their standard errors (SE) were calculated and
compared. Although the brood size in P. clarkii
ranges from dozens to hundreds (Trimble & Gaude
1988), the average GSI (gonadosomatic index,
100W
ovary
/(W
body
)=GSI%), average HSI (hepatoso-
matic index, 100W
ovary
/W
body
=HSI%) and the
average oocyte diameter (OD) in each group were
determined for reference.
Eyestalk interventional injection
The interventional agent (gelatin containing
0.1 g mL
1
TRA, 2 mg mL
1
DOM or 2 mg mL
1
5-HT) was injected into the internal base of the
eyestalk (proximal medulla terminalis, see Fig. 1)
of 135 females (45 females for each agent) via a
1-mL insulin syringe with a sterilized, permanently
attached, 29G91/2 inch needle (BD Ultra-Fine,
USA). The injection volume was 0.20.3 mL per
eyestalk.
Eyestalk ablation
Using a sterilized scalpel, 45 females had both eye-
stalks ablated (BEA) and 45 females had one eye-
stalk ablated (UEA) at the proximal end. Following
the application of powdered, hot and dry BSP (kept
at 75~80C before use), the specimens were placed
2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 14021414 1405
Aquaculture Research, 2014, 45, 14021414 Eyestalk intervention for maturation induction S Liu et al.
in water until the wound healed. The scalpel was
sterilized by dipping in 70% alcohol after each
craysh was treated.
Abdominal injection of DOM with or without 5-HT
Forty-ve females were injected in the second abdomi-
nal segment with 0.25 mL 2 mg mL
1
domperidone
solution (0.5 mg craysh
1
, about 1720 lg g
1
b.w.). In addition, 45 females were injected in the sec-
ond abdominal segment with 0.25 mL 2 mg mL
1
domperidone solution and 0.25 mL 2 mg mL
1
5-HT
solution (0.5 mg craysh
1
, about 1720 lg g
1
b.w.). The craysh were injected at 3-day intervals.
Sample processes and ovarian development stages
register
The sampling process consisted of two steps: First,
females which had completed spawning and dead
individuals were counted and removed each day.
Spawning craysh were calculated as ovary-
mature. Second, at the end of the experiment, the
ovaries in surviving individuals which had not
spawned were dissected. Ovarian development
stages were registered based on external observa-
tion of ovarian colour and oocyte size (Table 1) as
described previously by Li and Z. (1999), Ando
and Makioka (1998) and Kulkarni, Glade and Fin-
german (1991a). As the mature oocytes of
P. clarkii were very large (diameter often exceeded
1.4 mm) and it was difcult to obtain histological
sections, oocyte diameter (OD) was measured
under a Leica MZ7.5 high-performance stereomi-
croscope in 4% formalin using an ocular microme-
tre. Twenty-ve advanced-stage oocytes were
measured in each ovary. The remainder of the
ovary was xed in Bouins solution to obtain histo-
logical sections for the conrmation of OD. Previ-
tellogenic/primary oocytes were not used for OD
measurement as they reduced to a very small pro-
portion (<5%) in the ovary of P. clarkii since ovar-
ian stage III (Fig. 2P). Figure 2 shows the
characteristics of the different ovarian development
stages in P. clarkii.
Figure 1 The intervention agent was injected into the
inside base of the eyestalk via 1ml insulin syringe with
Permanently Attached 29G x 1/2 in. BD Ultra-Fine
Needle (U-100).
Table 1 Ovarian development stages, oocyte steps and gross appearance of ovary in red swamp craysh Procambarus
clarkii
Ovarian stages Histology Gross appearance OD (lm)
I Oogonia and very
early previtellogenic
oocytes
Fig. 2J White or translucent
thin sac, distinguishable
oocytes with large nucleus
or undistinguishable, Fig. 2A
Early previtellogenesis
(Immature stage oocytes)
< 30
II Preliminary
vitellogenesis
Fig. 2K White, yellowish or yellow
oocytes, Fig. 2B,C
Vitellogenic oocytes 10~300
III Primary
vitellogenesis
Fig. 2L,M,P Yellow and khaki oocytes,
Fig. 2C,D,E,H
250~500
IV Secondary
vitellogenesis
Fig. 2N Khaki and brown oocytes,
Fig. 2E,G
450~1600
V Maturation Fig. 2O Dark brown oocytes,
Fig. 2G
Maturation & reabsorption >1400
Figure 2A to P represent the gross appearance and histological changes during ovarian maturation; OD represents oocyte diameter;
Ovarian development stages in P. clarkii were classied based on the external observation of ovarian colour and oocyte diameter as
described previously (Ando & Makioka 1998; Kulkarni et al. 1991a, b; Li & Z. 1999) with overall consideration and slight modica-
tion. In this table, the histological picture of ovarian stage V, Fig. 2O, was obtained using an ovary, which was very close to com-
pletely maturation, as it is very difcult to obtain histological sections using large, completely mature oocytes of P. clarkii.
2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 14021414 1406
Eyestalk intervention for maturation induction S Liu et al. Aquaculture Research, 2014, 45, 14021414
Statistical analyses
The values of survival rate (SR,%) and synchronous
maturation rate (SMR,%) represent as Mean SE
(standard errors of the 3 parallel aquariums in each
group,%). The average gonadosomatic index (GSI,%),
average hepatosomatic index (HSI, I%) and the
average oocyte diameter (OD) represent as Mean
SD. The data were analysed with the Sigmaplot.13
program using one-way analysis of variance (ANO-
VA) and Ducans multiple range test. A difference
was considered to be signicant at P < 0.05.
Results
Synchronous ovarian maturation rate
Bilateral eyestalk ablation and unilateral eyestalk
ablation
Bilateral eyestalk ablation (BEA) signicantly pro-
moted ovarian maturation in P. clarkii (P < 0.05).
When spawning occurred in the BEA group, most
females in the group matured synchronously. A
signicantly higher SMR (88.89 11.11) was
detected when compared with the control group
(22.05 3.06). Unilateral eyestalk ablation (UEA)
also induced SMR when compared with the control
group (P < 0.05), but was not statistically signicant
in BEA (P > 0.05). In general, SMR in UEA craysh
(63.33 18.56) was lower than that in BEA craysh
and higher than that in the control group (Fig. 3).
Eyestalk interventional injections
In the eyestalk interventional injection groups, the
SMR of the EI-TRA group (37.78 2.22) was
lower than that in the UEA, EI-DOM
(66.67 9.62) and the combined abdominal
injection with DOM and 5-HT (AI-DOM+5-HT)
groups (P > 0.05), but higher than the EI-5-HT
(22.22 11.11) and control groups (P > 0.05).
The SMR of the EI-DOM group was signicantly
higher than the EI-5-HT and control groups
(P < 0.05), and was similar to the UEA and
AI-DOM+5-HT groups (P > 0.05). However, the
EI-DOM group also exhibited a lower SMR when
compared with the BEA group (P > 0.05). The
SMR in the EI-5-HT group was similar to that in
the control group (P > 0.05) (Fig. 3).
Abdomen injection of DOM with or without 5-HT
The abdominal injection with combined DOM and
5-HT (AI-DOM+5-HT) resulted in a lower SMR
(A) (B) (C)
(E) (F)
(H) (I)
(J) (K)
(L) (M)
(N)
(P)
(O)
(G)
(D)
Figure 2 The gross appearance and histological char-
acter of oocytes during the ovarian development of red
swamp craysh P. clarkii. Picture A~H show the gross
appearance changes during ovarian maturation and
Picture J~P show the histological changes during ovar-
ian stage I~IV; Oog represents oogonia; Oc1, Oc2, Oc3,
Oc4, mOc represent the 5 stages of oocytes. Fig.2I
shows an ovigerous female carrying big eggs (diameter
reached 2.30 mm) obtained in this study.
2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 14021414 1407
Aquaculture Research, 2014, 45, 14021414 Eyestalk intervention for maturation induction S Liu et al.
(59.26 10.14) when compared with the BEA
and UEA groups (P > 0.05), but showed a much
higher SMR than the control group (P < 0.05).
However, abdominal injection with DOM solution
alone (AI-DOM) achieved a SMR of 22.69 2.52,
similar to the control group (P > 0.05) (Fig. 3).
Survival rate
Bilateral eyestalk ablation and unilateral eyestalk
ablation
Both bilateral eyestalk ablation and unilateral eye-
stalk ablation led to high mortality in P. clarkii
(P < 0.05). Moreover, in the 32-day experiment
(90 samples), we detected no signicant difference
between the SR of the BEA (15.56 2.22) and
UEA (24.44 5.88) groups (P > 0.05) (Fig. 3).
Eyestalk interventional injections
The eyestalk interventional injection groups EI-TRA
(28.89 4.44) and EI-5-HT (15.56 4.44) had a
much lower SR than the control group
(71.11 5.88) and the AI-DOM group
(77.78 5.88) (P < 0.05).Compared with the BEA
and UEA groups, there was no signicant increase in
SR (P > 0.05) in the EI-TRA and EI-5-HT groups.
However, EI-DOM showed a SR (53.33 3.85) sig-
nicantly higher than those of the BEA and UEA
groups (P < 0.05), but lower than the AI-DOM and
control groups (71.11 5.88) (P < 0.05) (Fig. 3).
Abdomen injection of DOM with or without 5-HT
The combined injection of DOM and 5-HT
(AI-DOM+5-HT) resulted in a relatively high SR of
51.11 4.44, which was signicantly higher
than that in the eyestalk ablation groups
(P < 0.05), but lower than the control group
(71.11 5.88) (P < 0.05). However, injection of
DOM (AI-DOM) resulted in a higher SR than the
control group (P > 0.05) (Fig. 3).
GSI, HSI and OD of different groups
Table 2 shows the GSI (MeanSD,%), HSI
(Mean sd,%) and OD (Mean sd) of each
group when the specimens were dissected at the
end of the experiment. The GSI was highest in
the BEA (4.33 2.10) and EI-DOM
(3.26 2.15) groups, followed by the UEA
(2.97 2.61), EI-TRA (1.76 2.00), AI-
DOM+5-HT (1.62 1.10), control (1.24 1.68),
EI-5-HT (1.21 2.24) and AI-DOM (1.17 1.49)
groups. The GSI in the EI-DOM group was lower
than that in the BEA group (P > 0.05), but sig-
nicantly higher than that in the control group
and the abdominal injection groups AI-DOM and
AI-DOM+5-HT (P < 0.05). The GSI in the EI-
DOM group was lower than that in the UEA
group (P > 0.05), but higher than that in the AI-
DOM+5-HT, AI-DOM and the control groups
(P > 0.05). The GSI in the EI-5-HT group was
lower than that in the control group (P > 0.05),
but higher than that in the AI-DOM group
(P > 0.05). The HSI was lowest in the BEA
(4.46 1.08), EI-TRA (5.59 1.64) and
EI-DOM (5.53 1.58) groups, followed by the
UEA (6.37 1.63), control (6.54 1.22),
Figure 3 The survival rate (SR) and synchronous
ovarian maturation rate (SMR) of Procambrus clarkii
performed different treatments (sampled when two
ovigerous females were observed in the bilateral eyestalk
ablation group). BEA: bilateral eyestalk ablation; UEA:
unilateral eyestalk ablation; EI-TRA: eyestalk interven-
tion with TRA; EI-DOM: eyestalk intervention with
DOM; EI-5-HT: eyestalk intervention with 5-HT;
AI-DOM+5HT: combined injection with 2mgml-1 5-HT
and 2mgml-1 DOM solution (0.5 mg/craysh of each
agent) in the abdomen in 3-day intervals; AI-DOM:
injection with 2mgml-1 DOM solution (0.5 mg/cray-
sh) in the abdomen in 3-day intervals. Control: con-
trol group, untreated. SR and SMR were expressed as
Mean SE (%) in the gure. Statistical analyses used
the Sigmaplot.13 program using one-way analysis of
variance (ANOVA) and Ducans multiple range test.
A difference was considered to be signicant at P < 0.05.
2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 14021414 1408
Eyestalk intervention for maturation induction S Liu et al. Aquaculture Research, 2014, 45, 14021414
AI-DOM+5-HT (6.63 1.15), AI-DOM
(6.99 1.24) and EI-5-HT (7.31 1.20) groups.
The eyestalk interventional injection groups, EI-
TRA and EI-DOM, exhibited a higher HSI than
the BEA group (P > 0.05), but lower than the
UEA (P > 0.05) and the control groups
(P < 0.05). The HSI in the EI-5-HT, AI-DOM+5-
HT and AI-DOM groups was even higher than
the control group (P > 0.05). The OD was high-
est in the EI-DOM (1478 616) group, followed
by the BEA (1443 311), AI-DOM+5-HT
(1415 612), AI-DOM (1256 634), UEA
(1200 454), EI-TRA (1038 536), control
(1011 622) and
EI-5-HT (820 405) groups. The eyestalk inter-
ventional injection group, EI-DOM, had a higher
OD than the control group (P < 0.05), the eye-
stalk ablation groups, BEA and UEA (P > 0.05),
the abdominal injection groups, AI-DOM+5-HT
and AI-DOM (P > 0.05), and the eyestalk inter-
ventional injection group EI-TRA (P > 0.05). The
OD in the EI-5-HT group was not signicantly
higher than the control group (P > 0.05).
Discussion
This study, for the rst time and without prece-
dent, documented the results of developing an eye-
stalk interventional injection method to induce
synchronous ovarian maturation in P. clarkii and
compared the application values of safety and ef-
ciency to the effective traditional eyestalk ablation
method and the possible effective method of com-
bined injection with 5-HT and DOM.
Our experiment was performed on females of
advanced ovarian stages (ovarian stage III and
IV), as eyestalk ablations are seldom performed in
females of earlier ovarian stages in aqua farming,
because of its low application value. In addition,
eyestalk ablation might not lead to accelerated
ovarian maturation in immature P. clarkii, as Var-
alakshmi and Reddy (2010) reported that both
unilateral and bilateral eyestalk ablation failed to
induce accelerated ovarian maturation in the
prawn M. lanchesteri at an earlier ovarian stage.
No male P. clarkii were used in our experiment for
the following reasons: First, it is very difcult to
maintain a constant ratio of males to females
among different treatments as the mortality rate
varies with treatments. Even if we adjusted the
number of males according to the survival rate of
females to maintain a sex ratio of 1:1, there are
difculties in determining whether males are
mature. Second, one of the mating peaks in
P. clarkii in the Wuhan area occurs during June to
August, just before we selected the females for this
experiment, and the females carry long-term active
spermatophores after mating, as we have previ-
ously observed and reported (Jianlin 2006). Hence,
we believe that the number of females carrying
spermatophores was constant in the different
treatments when they were randomly assigned to
these treatments. The majority of the females
(88.89 11.11%) in the bilateral eyestalk ablation
Table 2 The gonadosomatic index (GSI), hepatosomatic index (HSI) and oocyte diameter (OD) of craysh Procambarus
clarkii in each group at the end of the experiment
Group Initial N
Survival Maturation
GSI HSI OD (lm)
N R (%) N R (%) (mean sd%) (mean SD%) (mean sd%)
A1 45 7 15.56 6 85.71 4.33 2.10
a
4.46 1.08
b
1443 311
ab
A2 45 11 24.44 6 54.55 2.97 2.61
ab
6.37 1.63
ab
1200 454
ab
B1 45 13 28.89 5 38.46 1.76 2.00
b
5.53 1.58
b
1038 536
ab
B2 45 23 51.11 15 65.22 3.26 2.15
a
5.59 1.64
b
1478 616
a
B3 45 7 15.56 1 15.56 1.21 2.24
b
7.31 1.20
a
820 405
b
C1 45 23 51.11 14 60.87 1.62 1.10
b
6.63 1.15
a
1415 612
ab
C2 45 35 77.78 8 22.69 1.17 1.49
b
6.99 1.24
a
1256 634
ab
Control 45 32 71.11 7 22.05 1.24 1.68
b
6.54 1.22
a
1011 622
b
N represents the number of specimens and R represents rate (%). GSI and HSI were calculated using the following equations:
100W
ovary
/(W
body
) = GSI% and 100W
ovary
/W
body
= HSI%. Previtellogenic/primary oocytes were not used for OD measurement as
they reduced to a very small proportion in the ovary of P. clarkii since ovarian stage III (see Fig. 2P); Statistical analyses included
the Sigmaplot.13 program using one-way analysis of variance (ANOVA) and Duncans multiple range test. A difference was consid-
ered to be signicant at P < 0.05. Experimental groups were named A1(BEA), A2(UEA), B1(EI-TRA), B2(EI-DOM), B3(EI-5-HT), C1
(AI-DOM+5-HT) and C2(AI-DOM) in the Table.
2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 14021414 1409
Aquaculture Research, 2014, 45, 14021414 Eyestalk intervention for maturation induction S Liu et al.
group in this study matured synchronously when
two ovigerous females were observed. This sug-
gested that the majority of the females we selected
in late August had mated and carried active sper-
matophores. Third, recent research suggests that
P. clarkii may also be reproduced by parthenogene-
sis (Yue, Wang, Zhu, Wang, Zhu & Lo 2008; Li,
Deng, Yang & Wang 2012).
In this study, our experiment using P. clarkii
was carried out over a prolonged period of
32 days. The start time in this ovarian induction
experiment was limited by the need to select speci-
mens carrying active spermatophores. The timing
to close the experiment was decided, for the rst
time by us, when at least two ovigerous females
were observed in the bilateral eyestalk ablation
group, which required 32 days. We believe that
this is the preferred method to decide the time
interval before starting this type of ovarian induc-
tion experiment when a new method or dose of
agent is compared with a proved effective method
or dose of agent. In several previous studies, the
evaluation of ovarian maturation induction
depended on comparing the number of days before
spawning, which was known as the ovarian matu-
ration period (OMP) (Tinikul, Soonthornsumrith,
Phoungpetchara, Meeratana, Poljaroen, Duangsu-
wan, Soonklang, Mercier & Sobhon 2009), or the
periodically average maturation index (M.I.)
(Alfaro et al. 2004), or weekly spawning rate
(Vaca & Alfaro 2000) as well as the mean GSI
(Radhakrishnan & Vijayakumaran 2011) or Vg
levels (Tinikul et al. 2008) in a given period. These
given periods seem subjective, and lack a uniform
standard. In fact, a comparison of synchronous
maturation rate (SMR) when the method is known
to be effective is preferred to evaluate the effect of
different treatments on ovarian maturation induc-
tion efciency in P. clarkii, as the P. clarkii speci-
mens were mostly obtained from natural ponds
and it was very difcult to select specimens at the
same ovarian stage. Although a study on the cray-
sh Cherax quadricarinatus detected Vg levels
during its reproductive cycle (Ferre, Medesani,
Garca, Grodzielski & Rodrguez 2012), we did not
include this subject in our study. A comparison of
Vg levels has been reported in prawns such as M.
rosenbergii (Chen et al. 2003; Tinikul et al. 2008),
but has not been reported in the craysh P. clarkii.
Moreover, achieving synchronous ovarian matura-
tion is enough to break the production bottleneck
of P. clarkii juveniles for use in aquaculture. In this
study, we also determined the GSI, HSI and OD in
different treatments to support the calculated SMR,
although the brood size of P. clarkii ranges from
dozens to hundreds (Trimble & Gaude 1988) and
the OD of spawned eggs in different females ranged
from 1.1 to 2.3 mm (Fig. 2I).
As expected, with mature female P. clarkii, both
bilateral and unilateral eyestalk ablation resulted
in a signicantly higher SMR (P < 0.05) and led
to a much higher mortality compared with the
control (P < 0.05). The maturation induction
results were consistent with the ndings following
scalding of the eyestalks in P. clarkii (Ruijie 2009),
and the mortality rate was consistent, but lower,
with the trend reported in M. lanchesteri (Var-
alakshmi & Reddy 2010). Compared with eyestalk
ablation, abdominal injection of a proved effective
dosage, 0.5 mg craysh
1
(0.5 mg craysh
1
= 17
20 lg g
1
b.w. = 2.5 9 10
6
mol craysh
1
in
this study), of 5-HT combined with DOM, resulted
in a signicantly lower SMR (P > 0.05), but much
higher SR (P < 0.05). Abdominal injection of DOM
alone failed to induce accelerated ovarian matura-
tion and only achieved an SMR non-signicantly
higher than the untreated control (P > 0.05).
Such results are consistent with the reports by
Chen et al. (2003) using M. rosenbergii and Alfaro
et al. (2004) using L. stylirostris and L. vannamei.
As a D2-type DA antagonist, DOM injection alone,
at a dosage of 1720 lg g
1
b.w., seemed to have
limited effectiveness. However, DOM seemed to
increase the SR of P. clarkii, as the SR following
DOM injection was even higher than that in the
control group (P > 0.05), which, to our know-
ledge, has not previously been demonstrated.
Unlike DOM injection alone, eyestalk interven-
tional injection of DOM resulted in a signicantly
higher SMR than the control (P < 0.05), but was
non-signicantly lower than eyestalk ablation and
combined injections of 5-HT and DOM (P > 0.05).
A comparison of GSI, HSI and OD among the
treatments supported the above results. Eyestalk
interventional injection of DOM also led to a better
SR, which was signicantly higher than the eye-
stalk ablations (P < 0.05). Although the SR was
still lower than that in the control group
(P < 0.05), no signicant difference was observed
when it was compared with that in the combined
injection of 5-HT and DOM group (P > 0.05).
These results suggest that eyestalk interventional
injection of DOM has the positive characteristics of
a relatively low mortality rate and higher SMR
2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 14021414 1410
Eyestalk intervention for maturation induction S Liu et al. Aquaculture Research, 2014, 45, 14021414
and may be a potential technique for inducing
ovarian maturation in P. clarkii as an alternative
technique to traditional eyestalk ablation. The
mechanism to explain the signicantly higher
SMR (P < 0.05) achieved in the DOM eyestalk
interventional group compared with the DOM
abdominal injection group deserves further study.
We agree that DA is a crucial neurotransmitter,
which inhibits ovarian maturation (Chen et al.
2003); however, we think that the role of eye-
stalks in the effective changes in DA level which
control ovarian development is more important
than other tissues such as the thoracic ganglia,
which was also proposed by Chen et al. (2003).
Disappointingly, eyestalk interventional injection
of TRA resulted in a SMR only slightly higher
than that in the control group (P > 0.05), but
was still lower than that in the BEA (P < 0.05),
UEA (P > 0.05), as well as the combined injection
of 5-HT and DOM groups (P > 0.05). It also
resulted in a SR signicantly lower than that in
the control group and the combined injection of
5-HT and DOM group (P < 0.05), but was slightly
higher than both the BEA and UEA groups
(P > 0.05) (Fig. 3). In addition, the levels of OD
and GSI were non-signicantly higher than the
control (P > 0.05), whereas the level of HSI exhib-
ited a signicant decreasing trend compared with
the control group (P < 0.05) and was non-signi-
cantly higher than that in the BEA group
(P > 0.05). These results failed to provide new evi-
dence to support the most commonly accepted
theory, which proposed that the sinus gland (SG)
in the eyestalks is the storage and release site of
the haemolymph-borne neurohormones, such as
GIH and MOIH, which inhibit precocious ovarian
maturation in non-breeding seasons (Bray & Law-
rence 1992). Such a vague effect on accelerating
ovarian maturation can be explained by the fact
that eyestalk intervention with gelatin containing
TRA failed to embolize the eyestalk to prevent GIH
and MOIH release into the haemolymph circula-
tion. Another explanation is that the eyestalk tis-
sue does not play a signicant role in releasing
haemolymph-borne GIH or MOIH to control ovar-
ian maturation; thus even when both GIH and
MOIH release from the eyestalks was effectively
blocked by the gelatin containing TRA injection,
other inhibiting factors outside the eyestalks pre-
vented precocious ovarian maturation in P. clarkii.
Several previous studies seem to support the latter
explanation. Chen et al. (2003) reported that DA
was able to inhibit Vg synthesis in eyestalk-
ablated M. rosenbergii and proposed that DA exerts
its inhibitory effect on Vg synthesis by inhibiting
the release of GSH from the thoracic ganglia
rather than stimulating the release of GIH from
the eyestalks. Alvarez et al. (2005) reported that
the tissue distribution of DA in P. clarkii was not
limited to the eyestalks. In our opinion, further
study such as examining the changes in DA,
MOIH, GIH as well as GSH levels after eyestalk
ablation, eyestalk embolization and during ovarian
maturation may cast light on the mechanism
involved.
Unexpectedly and unsatisfactorily, eyestalk
interventional injection of 5-HT achieved the
worst results among all the treatments. It showed
a similar SMR to that of the control (P > 0.05), a
similar SR to the BEA group (P > 0.05) and a sig-
nicantly lower SR than the control (P < 0.05).
These negative results were unexpected as we
achieved signicantly higher SMR, GSI and OD fol-
lowing abdominal injection of DOM with 5-HT
compared with that without 5-HT. 5-HT was also
reported to extensively accelerate ovarian matura-
tion in previous studies in the craysh P. clarkii
(Sarojini et al. 1995b; Kulkarni et al. 1992) as
well as the shrimp L. vannamei (Vaca & Alfaro
2000), F. merguiensis (Zacharia & Kakati 2011),
the prawn M. rosenbergii (Tinikul et al. 2009) and
several other decapods. Fingerman (1997) and
Kulkarni et al. (1992) suggested that 5-HT might
act indirectly on the gonads by stimulating the
release of a putative gonadotropic factor such as
GSH from the thoracic ganglia, and/or by inhibit-
ing the release of GIH from the optic lobe in the
eyestalk of P. clarkii. The primary target of 5-HT
seems to be the X-organ neurons in the eyestalks
(Tinikul et al. 2009). The mechanism to explain
the difference between the ndings following 5-HT
injection and those following the 5-HT eyestalk in-
terventional injection in this report also deserves
further study. From our current research, we are
unable to provide a convincing explanation for the
unsatisfactory effect of the eyestalk interventional
injection of 5-HT on the induction of ovarian mat-
uration. However, as we were concerned with the
safety aspects when we evaluated the efciency of
5-HT on inducing ovarian maturation, one point
we would like to stress is that 5-HT appears to
increase the mortality rate of P. clarkii (Fig. 3),
which, to our knowledge, has not previously been
demonstrated.
2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 14021414 1411
Aquaculture Research, 2014, 45, 14021414 Eyestalk intervention for maturation induction S Liu et al.
There is no precedent, in prior studies, to follow
in performing an eyestalk intervention method.
For this study, we selected the BSP gelatin to make
the carrier for TRA, 5-HT or DOM because it is a
biological material that is non-dispersible, sus-
tained-releasing and harmless. Feng et al. (1996)
reported its character of non-dispersiblity. Tradi-
tional Chinese medicine has the principle of not
using Bletilla striata with aconite root because it
prolongs the time of aconite root toxicity (Yeung
1985) suggesting that it is a sustained-releasing
material. As an internal medicine for humans, it is
also harmless. Hence, we believe that BSP is basi-
cally a safe and neutral vector for interventional
injection. In this study, all interventional agents
were made of the crude BSP we extracted; the
survival rate and ovarian maturation induction
performance seems to relate to the TRA, DOM or
5-HT; it contains rather than BSP itself. It deserves
further tests and improvement to be applicable to
mass production of interventional injection agents.
In summary, our investigation into the induc-
tion of ovarian maturation in P. clarkii via eyestalk
interventional injection was based on the hypothe-
sis that the eyestalk plays an incomparable role in
releasing neuropeptides such as haemolymph-
borne GIH and MOIH, and is deeply involved in
the antagonistic action of DA and 5-HT. We
induced ovarian maturation by disturbing the eye-
stalks functions via interventional injections of a
gelatin containing TRA, DOM or 5-HT into the
eyestalk of P. clarkii. Our ndings appear to show
that the eyestalk is deeply involved in the inhibi-
tory action of DA during the ovarian maturation
in P. clarkii. In practice, the positive characteristics
of eyestalk interventional injection with DOM have
implications in the design of a safe, efcient, inex-
pensive, less labour-intensive and time-controllable
method to induce ovarian maturation in decapods.
The traditional eyestalk ablation technique is
cruel, the speed of maturation is uncontrollable,
and leads to permanent damage, whereas injection
of 5-HT and/or a DA antagonist needs to be per-
formed repeatedly and is expensive. These methods
are not widely adopted in the mass production of
stocking seeds in the inexpensive craysh P. clarkii.
In this study, eyestalk interventional injection was
only performed once, and BSP, which is an abun-
dant material in nature, deserves further assess-
ment as a safe, efcient, inexpensive and less
labour-intensive method for maturation induction
in decapods. Moreover, by regulating the dosage of
the interventional injection agent, it is possible to
control the speed of ovarian maturation induction.
Eyestalk intervention methods also provide other
ways to explore the neuroendocrine function of
the XO-SG of these species in vivo. In the prior
studies, the eyestalk tissue was ablated or
implanted into the abdomen to note or measure its
effect or response.
Acknowledgments
We are grateful to Professor W. Zhao of Shanghai
Ocean University, Professor W. Wang, senior engi-
neer M. Wang and Dr. Z. Luo of Huazhong Agri-
cultural University for their contributions and
assistance. This research was supported by the
program Transformation of Agricultural Science
and Technology Achievements (project number
4002-092062) nanced by the ministry of nance
china.
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